Methods and products for analyzing polymers

ABSTRACT

Methods and products for analyzing polymers are provided. The methods include methods for determining various other structural properties of the polymers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/432,959 filed Mar. 28, 2012, which is a continuation of U.S.application Ser. No. 09/852,968 filed May 10, 2001, which is a divisionof U.S. application Ser. No. 09/134,411 filed Aug. 13, 1998, now U.S.Pat. No. 6,355,420, which is a continuation of International applicationno. PCT/US 1998/003024, filed Feb. 11, 1998, which claims priority toU.S. provisional application Nos. 60/037,921 filed Feb. 12, 1997 and60/064,687 filed Nov. 5, 1997, which disclosures are herein incorporatedby reference in their entirety.

REFERENCE TO BIOLOGICAL SEQUENCE DISCLOSURE

This application contains nucleotide sequence and/or amino acid sequencedisclosure in computer readable form and a written sequence listing, theentire contents of both of which are expressly incorporated by referencein their entirety as though fully set forth herein.

BACKGROUND

The study of molecular and cellular biology is focused on themacroscopic structure of cells. We now know that cells have a complexmicrostructure that determine the functionality of the cell. Much of thediversity associated with cellular structure and function is due to theability of a cell to assemble various building blocks into diversechemical compounds. The cell accomplishes this task by assemblingpolymers from a limited set of building blocks referred to as monomers.The key to the diverse functionality of polymers is based in the primarysequence of the monomers within the polymer and is integral tounderstanding the basis for cellular function, such as why a celldifferentiates in a particular manner or how a cell will respond totreatment with a particular drug.

The ability to identify the structure of polymers by identifying theirsequence of monomers is integral to the understanding of each activecomponent and the role that component plays within a cell. Bydetermining the sequences of polymers it is possible to generateexpression maps, to determine what proteins are expressed, to understandwhere mutations occur in a disease state, and to determine whether apolysaccharide has better function or loses function when a particularmonomer is absent or mutated.

Expression maps relate to determining mRNA expression patterns. The needto identify differentially expressed mRNAs is critical in theunderstanding of genetic programming, both temporally and spatially.Different genes are turned on and off during the temporal course of anorganisms' life development, comprising embryonic, growth, and agingstages. In addition to developmental changes, there are also temporalchanges in response to varying stimuli such as injury, drugs, foreignbodies, and stress. The ability to chart expression changes for specificsets of cells in time either in response to stimuli or in growth allowsthe generation of what are called temporal expression maps. On the otherhand, there are also body expression maps, which include knowledge ofdifferentially expressed genes for different tissues and cell types.Expression maps are different not only between species and betweenindividuals, but also between diseased and disease-free states.Examination of differential gene expression has yielded key discoveriesof genes in widely varying disciplines, such as signal transduction(Smith et al., 1990), circadian rhythms (Loros et al., 1989), fruitripening (Wilinson et al., 1995), hunger (Qu et al., 1996), cell cyclecontrol (el-Deiry et al., 1993), apoptosis (Woronicz et al., 1994), andischemic injury (Wang et al., 1995), among many others. Since generationof expression maps involve the sequencing and identification of cDNA ormRNA, more rapid sequencing necessarily means more rapid generation ofmultiple expression maps.

Currently, only 1% of the human genome and an even smaller amount ofother genomes have been sequenced. In addition, only one very incompletehuman body expression map using expressed sequence tags has beenachieved (Adams et al., 1995). Current protocols for genomic sequencingare slow and involve laborious steps such as cloning, generation ofgenomic libraries, colony picking, and sequencing. The time to createeven one partial genomic library is on the order of several months. Evenafter the establishment of libraries, there are time lags in thepreparation of DNA for sequencing and the running of actual sequencingsteps. Given the multiplicative effect of these unfavorable facts, it isevident that the sequencing of even one genome requires an enormousinvestment of money, time, and effort.

In general DNA sequencing is currently performed using one of twomethods. The first and more popular method is the dideoxy chaintermination method described by Sanger et al. (1977). This methodinvolves the enzymatic synthesis of DNA molecules terminating indideoxynucleotides. By using the four ddNTPs, a population of moleculesterminating at each position of the target DNA can be synthesized.Subsequent analysis yields information on the length of the DNAmolecules and the base at which each molecule terminates (either A, C,G, or T). With this information, the DNA sequence can be determined. Thesecond method is Maxam and Gilbert sequencing (Maxam and Gilbert, 1977),which uses chemical degradation to generate a population of moleculesdegraded at certain positions of the target DNA. With knowledge of thecleavage specificities of the chemical reactions and the lengths of thefragments, the DNA sequence is generated. Both methods rely onpolyacrylamide gel electrophoresis and photographic visualization of theradioactive DNA fragments. Each process takes about 1-3 days. The Sangersequencing reactions can only generate 300-800 bases in one run.

Methods to improve the output of sequence information using the Sangermethod also have been proposed. These Sanger-based methods includemultiplex sequencing, capillary gel electrophoresis, and automated gelelectrophoresis. Recently, there has also been increasing interest indeveloping Sanger independent methods as well. Sanger independentmethods use a completely different methodology to realize the baseinformation. This category contains the most novel techniques, whichinclude scanning electron microscopy (STM), mass spectrometry, enzymaticluminometric inorganic pyrophosphate detection assay (ELIDA) sequencing,exonuclease sequencing, and sequencing by hybridization. A brief summaryof these methods is set forth below.

Currently, automated gel electrophoresis is the most widely used methodof large-scale sequencing. Automation requires reading of fluorescentlylabeled Sanger fragments in real time with a charge coupled device (CCD)detector. The four different dideoxy chain termination reactions are runwith different labeled primers. The reaction mixtures are combined andco-electrophoresed down a slab of polyacrylamide. Using laser excitationat the end of the gel, the separated DNA fragments are resolved and thesequence determined by computer. Many automated machines are availablecommercially, each employing different detection methods and labelingschemes. The most efficient of these is the Applied Biosystems Model377XL, which generates a maximum actual rate of 115,200 bases per day.

In the method of capillary gel-electrophoresis, reaction samples areanalyzed by small diameter, gel-filled capillaries. The small diameterof the capillaries (50 μm) allows for efficient dissipation of heatgenerated during electrophoresis. Thus, high field strengths can be usedwithout excessive Joule heating (400 V/m), lowering the separation timeto about 20 minutes per reaction run. Not only are the bases separatedmore rapidly, there is also increased resolution over conventional gelelectrophoresis. Furthermore, many capillaries are analyzed in parallel(Wooley and Mathies, 1995), allowing amplification of base informationgenerated (actual rate is equal to 200,000 bases/day). The main drawbackis that there is not continuous loading of the capillaries since a newgel-filled capillary tube must be prepared for each reaction. Capillarygel electrophoresis machines have recently been commercialized.

Multiplex sequencing is a method which more efficiently useselectrophoretic gels (Church and Kieffer-Higgins, 1988). Sanger reactionsamples are first tagged with unique oligomers and then up to 20different samples are run on one lane of the electrophoretic gel. Thesamples are then blotted onto a membrane. The membrane is thensequentially probed with oligomers that correspond to the tags on theSanger reaction samples. The membrane is washed and reprobedsuccessively until the sequences of all 20 samples are determined. Eventhough there is a substantial reduction in the number of gels run, thewashing and hybridizing steps are as equally laborious as runningelectrophoretic gels. The actual sequencing rate is comparable to thatof automated gel electrophoresis.

Sequencing by mass spectrometry was first introduced in the late 80's.Recent developments in the field have allowed for better sequencedetermination (Crain, 1990; Little et al., 1994; Keough et al., 1993;Smirnov et al., 1996). Mass spectrometry sequencing first entailscreating a population of nested DNA molecules that differ in length byone base. Subsequent analysis of the fragments is performed by massspectrometry. In one example, an exonuclease is used to partially digesta 33-mer (Smirnov, 1996). A population of molecules with similar 5′ endsand varying points of 3′ termination is generated. The reaction mixtureis then analyzed. The mass spectrometer is sensitive enough todistinguish mass differences between successive fragments, allowingsequence information to be generated.

Mass spectrometry sequencing is highly accurate, inexpensive, and rapidcompared to conventional methods. The major limitation, however, is thatthe read length is on the order of tens of bases. Even the best method,matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)mass spectroscopy (Smirnov et al., 1996), can only achieve maximum readlengths of 80-90 base pairs. Much longer read lengths are physicallyimpossible due to fragmentation of longer DNA at guanidines during theanalysis step. Mass spectrometry sequencing is thus limited to verifyingshort primer sequences and has no practical application in large-scalesequencing.

The Scanning tunneling microscope (STM) sequencing (Ferrell, 1997)method was conceived at the time the STM was commercially available. Theinitial promise of being able to read base-pair information directlyfrom the electron micrographs no longer holds true. DNA molecules mustbe placed on conducting surfaces, which are usually highly orderedpyrolytic graphite (HOPG) or gold. These lack the binding sites to holdDNA strongly enough to resist removal by the physical and electronicforces exerted by the tunneling tip. With difficulty, DNA molecules canbe electrostatically adhered to the surfaces. Even with successfulimmobilization of the DNA, it is difficult to distinguish baseinformation because of the extremely high resolutions needed. Withcurrent technology, purines can be distinguished from pyrimidines, butthe individual purines and pyrimidines cannot be identified. The abilityto achieve this feat requires electron microscopy to be able todistinguish between aldehyde and amine groups on the purines and thepresence or absence of methyl groups on the pyrimidines.

Enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA)sequencing uses the detection of pyrophosphate release from DNApolymerization to determine the addition of successive bases. Thepyrophosphate released by the DNA polymerization reaction is convertedto ATP by ATP sulfurylase and the ATP production is monitoredcontinuously by firefly luciferase. To determine base specificity, themethod uses successive washes of ATP, CTP, GTP, and TTP. If a wash forATP generates pyrophosphate, one or more adenines are incorporated. Thenumber of incorporated bases is directly proportional to the amount ofpyrophosphate generated. Enhancement of generated sequence informationcan be accomplished with parallel analysis of many ELIDA reactionssimultaneously.

The main disadvantage is the short read length. Ronaghi et al. (1996)have only achieved a maximum read length of 15 bases because of themultiple washings needed. Since there are four washes per base read,this means that a total of 400 washes mush be performed for a readlength of a hundred bases. If there is even 1% loss of starting materialfor each wash, after 400 washes there would be 1.8% of the startingmaterial remaining, which is insufficient for detection.

Exonuclease sequencing involves a fluorescently labeled, single-strandedDNA molecule which is suspended in a flowing stream and sequentiallycleaved by an exonuclease. Individual fluorescent bases are thenreleased and passed through a single molecule detection system. Thetemporal sequence of labeled nucleotide detection corresponds to thesequence of the DNA (Ambrose et al., 1993; Davis et al., 1992; Jett etal., 1989). Using a processive exonuclease, it theoretically is possibleto sequence 10,000 by or larger fragments at a rate of 10 bases persecond.

In practice, exonuclease sequencing has encountered many difficulties ineach of the steps. The labeling step requires that all four bases in theDNA be tagged with different fluorophores. Sterically, this is extremelyunfavorable. Ambrose et al., 1993 has achieved complete labeling of twobases on a 7 kb strand of M13 DNA. Furthermore, difficult opticaltrapping is needed to suspend DNA molecules in a flowing stream. Thestep is time intensive and requires considerable expertise. Lastly,single molecules of fluorophore need to be detected with highefficiency. Even a 1% error is significant. Improvements in detectionfrom 65% to 95% efficiency have been achieved. The efficiency ofdetection has been pushed to the limit and it would be difficult toachieve further improvements.

In the sequencing by hybridization method, a target DNA is sequentiallyprobed with a set of oligomers consisting of all the possible oligomersequences. The sequence of the target DNA is generated with knowledge ofthe hybridization patterns between the oligomers and the target (Bains,1991; Cantor et al., 1992; Drmanac et al., 1994). There are two possiblemethods of probing target DNA. The “Probe Up” method includesimmobilizing the target DNA on a substrate and probing successively witha set of oligomers. “Probe Down” on the other hand requires that a setof oligomers be immobilized on a substrate and hybridized with thetarget DNA. With the advent of the “DNA chip,” which applies microchipsynthesis techniques to DNA probes, arrays of thousands of different DNAprobes can be generated on a 1 cm² area, making Probe Down methods morepractical. Probe Up methods would require, for an 8-mer, 65,536successive probes and washings, which would take an enormous amount oftime. On the other hand, Probe Down hybridization generates data in afew seconds. With perfect hybridization, 65,536 octamer probes woulddetermine a maximum of 170 bases. With 65,536 “mixed” 11-mers, 700 basescan be generated.

In practice, Probe Up methods have been used to generate sequences ofabout 100 base pairs. Imperfect hybridization has led to difficulties ingenerating adequate sequence. Error in hybridization is amplified manytimes. A 1% error rate reduces the maximum length that can be sequencedby at least 10%. Thus if 1% of 65,536 oligonucleotides gave falsepositive hybridization signals when hybridizing to a 200-mer DNA target,75% of the scored “hybridizations” would be false (Bains, 1997).Sequence determination would be impossible in such an instance. Theconclusion is that hybridization must be extremely effective in order togenerate reasonable data. Furthermore, sequencing by hybridization alsoencounters problems when there are repeats in sequences that are onebase less than the length of the probe. When such sequences are present,multiple possible sequences are compatible with the hybridization data.

The most common limitation of most of these techniques is a short readlength. In practice a short read length means that additional geneticsequence information needs to be sequenced before the linear order of atarget DNA can be deciphered. The short fragments have to be bridgedtogether with additional overlapping fragments. Theoretically, with a500 base read length, a minimum of 9×10⁹ bases need to be sequencedbefore the linear sequence of all 3×10⁹ bases of the human genome areproperly ordered. In reality, the number of bases needed to generate abelievable genome is approximately 2×10¹⁰ bases. Comparisons of thedifferent techniques show that only the impractical exonucleasesequencing has the theoretical capability of long read lengths. Theother methods have short theoretical read lengths and even shorterrealistic read lengths. To reduce the number of bases that need to besequenced, it is clear that the read length must be improved.

Protein sequencing generally involves chemically induced sequentialremoval and identification of the terminal amino acid residue, e.g., byEdman degradation. See Stryer, L., Biochemistry, W. H. Freeman and Co.,San Francisco (1981) pp. 24-27. Edman degradation requires that thepolypeptide have a free amino group which is reacted with anisothiocyanate. The isothiocyanate is typically phenyl isothiocyanate.The adduct intramolecularly reacts with the nearest backbone amide groupof the polymer thereby forming a five membered ring. This adductrearranges and the terminal amino acid residue is then cleaved usingstrong acid. The released phenylthiohydantoin (PTH) of the amino acid isidentified and the shortened polymer can undergo repeated cycles ofdegradation and analysis.

Further, several new methods have been described for carboxy terminalsequencing of polypeptides. See Inglis, A. S., Anal. Biochem. 195:183-96(1991). Carboxy terminal sequencing methods mimic Edman degradation butinvolve sequential degradation from the opposite end of the polymer. SeeInglis, A. S., Anal. Biochem. 195:183-96 (1991). Like Edman degradation,the carboxy-terminal sequencing methods involve chemically inducedsequential removal and identification of the terminal amino acidresidue.

More recently, polypeptide sequencing has been described by preparing anested set (sequence defining set) of polymer fragments followed by massanalysis. See Chait, B. T. et al., Science 257:1885-94 (1992). Sequenceis determined by comparing the relative mass difference betweenfragments with the known masses of the amino acid residues. Thoughformation of a nested (sequence defining) set of polymer fragments is arequirement of DNA sequencing, this method differs substantially fromthe conventional protein sequencing method consisting of sequentialremoval and identification of each residue. Although this method haspotential in practice it has encountered several problems and has notbeen demonstrated to be an effective method.

Each of the known methods for sequencing polymers has drawbacks. Forinstance most of the methods are slow and labor intensive. The gel basedDNA sequencing methods require approximately 1 to 3 days to identify thesequence of 300-800 units of a polymer. Methods such as massspectroscopy and ELIDA sequencing can only be performed on very shortpolymers.

An enormous need exists for de noveau polymer sequence determination.The rate of sequencing has limited the capability to generate multiplebody and temporal expression maps which would undoubtedly aid the rapiddetermination of complex genetic function. A need also exists forimproved methods for analyzing polymers in order to speed up the rate atwhich diagnosis of diseases and preparation of new medicines is carriedout.

SUMMARY OF THE INVENTION

The invention relates to new methods and products for analyzing polymersand in particular new methods and products useful for determining thesequence of polymers. The invention has surprising advantages over priorart methods used to sequence polymers. Prior to the present invention nomethod or combination of methods has come close to achieving the rate ofsequencing which the instant invention is capable of achieving. Usingthe methods of the invention the entire human genome can be sequencedseveral orders of magnitude faster than could be accomplished usingconventional technology. In addition to sequencing the entire genome,the methods and products of the invention can be used to createcomprehensive and multiple expression maps for developmental and diseaseprocesses. The ability to sequence an individual's genome and togenerate multiple expression maps will greatly enhance the ability todetermine the genetic basis of any phenotypic trait or disease process.

The method for analyzing polymers according to the invention is based onthe ability to examine each unit of a polymer individually. By examiningeach unit individually the type of unit and the position of the unit onthe backbone of the polymer can be identified. This can be accomplishedby positioning a unit at a station and examining a change which occurswhen that unit is proximate to the station. The change can arise as aresult of an interaction that occurs between the unit and the station ora partner and is specific for the particular unit. For instance if thepolymer is a nucleic acid molecule and a T is positioned in proximity toa station a change which is specific for a T occurs. If on the otherhand, a G is positioned in proximity to a station then a change which isspecific for a G will occur. The specific change which occurs depends onthe station used and the type of polymer being studied. For instance thechange may be an electromagnetic signal which arises as a result of theinteraction.

The methods of the invention broadly encompass two types of methods foranalyzing polymers by identifying a unit (or in some cases a group ofunits) within a polymer. The first type of method involves the analysisof at least a single polymer. An individual unit of the single polymerin one aspect is caused to interact with an agent such that a change,e.g., energy transfer or quenching occurs and produces a signal. Thesignal is indicative of the identity of the unit. In another aspect anindividual unit is exposed to a station resulting in a detectablephysical change to the unit or station. The change in the unit orstation produces a signal which can be detected and is characteristic ofthat particular unit. The second type of method involves the analysis ofa plurality of polymers. A unit of each of the plurality of polymers ispositioned at a station where an interaction can occur. The interactionis one which produces a polymer dependent impulse that specificallyidentifies the unit. The polymer dependent impulse may arise from, forexample, energy transfer, quenching, changes in conductance, mechanicalchanges, resistance changes, or any other physical change.

The proposed method for analyzing polymers is particularly useful fordetermining the sequence of units within a DNA molecule and caneliminate the need for generating genomic libraries, cloning, and colonypicking, all of which constitute lengthy pre-sequencing steps that aremajor limitations in current genomic-scale sequencing protocols. Themethods disclosed herein provide much longer read lengths than achievedby the prior art and a million-fold faster sequence reading. Theproposed read length is on the order of several hundred thousandnucleotides. This translates into significantly less need foroverlapping and redundant sequences, lowering the real amount of DNAthat needs to be sequenced before genome reconstruction is possible.

Methods for preparing polymers for analysis are also claimed herein. Thecombination of the long read length and the novel preparation methodsresults in a much more stream-lined and efficient process. Lastly, theactual time taken to read a given number of units of a polymer is amillion-fold more rapid than current methods because of the tremendousparallel amplification supplied by a novel apparatus also claimedherein, which is referred to as a nanochannel plate or a microchannelplate. The combination of all these factors translates into a method ofpolymer analysis including sequencing that will provide enormousadvances in the field of molecular and cell biology.

The ability to sequence polymers such as genomic DNA by the methodsdescribed in the instant invention will have tremendous implications inthe biomedical sciences. The recovery of genetic data at such a rapidpace will advance the Human Genome Project. The methods and products ofthe invention will allow the capability to prepare multiple expressionmaps for each individual, allowing complete human genetic programs to bedeciphered. The ability to compare pools of individual genetic data atone time will allow, for the first time, the ability to discover notonly single gene diseases with ease, but also complex multigenedisorders as rapidly as the DNA itself is sequenced.

In one aspect the invention is a method for analyzing a polymer oflinked units. The method involves the steps of exposing a plurality ofindividual units of a polymer to an agent selected from the groupconsisting of electromagnetic radiation, a quenching source and afluorescence excitation source, individual units interacting with theagent to produce a detectable signal, and detecting sequentially thesignals resulting from said interaction to analyze the polymer. In oneembodiment the signal is electromagnetic radiation. In anotherembodiment the agent is electromagnetic radiation. According to anembodiment of the invention individual units of the polymer are labeledwith a fluorophore.

The plurality of individual units of the polymer may be sequentiallyexposed to electromagnetic radiation by bringing the plurality ofindividual units in proximity to a light emissive compound and exposingthe light emissive compound to electromagnetic radiation, and whereinthe plurality of individual units of the polymer detectably affectemission of electromagnetic radiation from the light emissive compound.In another embodiment the plurality of individual units of the polymerare sequentially exposed to electromagnetic radiation, and wherein theelectromagnetic radiation detectably affects emission of electromagneticradiation from the plurality of individual units of the polymer toproduce the detectable signal.

According to another embodiment of the invention the method involves thestep of moving the polymer through a nanochannel in a wall material inorder to locate the detectable signal. The plurality of individual unitsof the polymer are sequentially exposed to the agent by moving thepolymer through a nanochannel in a wall material and exposing theplurality of individual units of the polymer to the agent at aninteraction station at the nanochannel. The agent can be attached to(embedded in, covalently attached to the surface of or coated on thesurface of) the wall material. In one embodiment the wall materialincludes a plurality of nanochannels, an interaction station at eachnanochannel, and a plurality of polymers is moved through saidnanochannel, only one polymer passes the interaction station at anygiven time (more than one polymer may be in a single nanochannel at agiven time as long as they do not overlap), and signals resulting fromthe interaction of individual units of the polymers and the agent at theinteraction stations are detected simultaneously. Preferably thenanochannel is fixed in the wall material.

The signals which are detected can be stored in a database for furtheranalysis. In one method of analysis these signals can be compared to apattern of signals from another polymer to determine the relatedness ofthe two polymers. Alternatively the detected signals can be compared toa known pattern of signals characteristic of a known polymer todetermine the relatedness of the polymer being analyzed to the knownpolymer. The analysis may also involve measuring the length of timeelapsed between detection of a first signal from the first unit and asecond signal from a second unit. In one embodiment the plurality ofindividual units are two units, a first unit at a first end of thepolymer and a second unit at an opposite second end of the polymer. Thetime elapsed between the sequential detection of signals may indicatethe distance between two units or the length of the polymer.

The polymer may be any type of polymer known in the art. In a preferredembodiment the polymer is selected from the group consisting of anucleic acid and a protein. In a more preferred embodiment the polymeris a nucleic acid.

The units of the polymer which interact with the agent to produce asignal are labeled. The units may be intrinsically labeled orextrinsically labeled. In one embodiment only a portion of the units ofthe polymer are labeled. In another embodiment all of the units arelabeled. In yet another embodiment at least two units of the polymer arelabeled differently so as to produce two different detectable signals.The units of the polymer may be labeled such that each unit or aspecified portion of the units is labeled or it may be randomly labeled.

In another embodiment the plurality of individual units of the polymerare exposed to at least two stations positioned in distinct regions ofthe channel, wherein the interaction between the units of the polymerand the at least two stations produces at least two signals.

In one embodiment the individual unit of the polymer is labeled withradiation and the signal is electromagnetic radiation in the form offluorescence.

In another embodiment the unit is exposed to the agent at a station.Preferably the station is a non-liquid material.

In yet another embodiment the plurality of individual units of thepolymer are exposed to at least two agents and the interaction betweenthe units of the polymer and the at least two agents produces at leasttwo signals. The at least two agents may be positioned in distinctregions of a channel through which the polymer passes. In one embodimentthe at least two signals are different signals. In another embodimentthe at least two signals are the same signals.

According to another aspect of the invention a method for analyzing apolymer of linked units is provided. The method involves the steps ofmoving a plurality of individual units of a polymer of linked units withrespect to a station and detecting sequentially signals arising from adetectable physical change in the polymer or the station as individualunits pass the station to analyze the polymer. This aspect of theinvention also encompasses each of the embodiments discussed above.

In one embodiment the station is an interaction station and theindividual units are exposed at the interaction station to an agent thatinteracts with the individual unit to produce a detectableelectromagnetic radiation signal characteristic of the interaction. Inanother embodiment the station is a signal generation station and thecharacteristic signal produced is a polymer dependent impulse.Preferably the station is a non-liquid material.

In another aspect the invention is a method for analyzing a polymer oflinked units by exposing a plurality of individual units of a polymer toa station to produce to produce a non-ion conductance signal resultingfrom the exposure of the units of the polymer to the station, andwherein the station is attached to a wall material having a surfacedefining a channel. This aspect of the invention also encompasses eachof the embodiments discussed above.

According to another aspect of the invention a method for identifying anindividual unit of a polymer is provided. The method involves the stepsof transiently exposing the individual unit of the polymer to an agentselected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source, the identity ofthe individual unit being unknown, to generate an interaction with adetectable electromagnetic radiation signal characteristic of saidindividual unit, detecting said signal, and distinguishing said signalfrom signals generated from adjacent signal generating units of thepolymer as an indication of the identity of the individual unit.

The agent can be one or more fluorophores and preferably is at leastthree fluorophores. When the individual unit is transiently exposed toone or more fluorophores (agent) by positioning the individual unitwithin energy transfer proximity of the agent, fluorescence energytransfer occurs between the agent and the individual unit. The signal isdetected by detecting the fluorescence energy transfer.

In one embodiment the individual unit of the polymer is exposed to theagent by positioning the individual unit at an interaction stationcomprising a nanochannel in a wall material. Preferably the wallmaterial comprises at least two layers, one of the layers allowingsignal generation and the other preventing signal generation and thenanochannel traverses both layers.

According to another aspect the invention is a method for identifying anindividual unit of a polymer. The method includes the steps oftransiently moving the individual unit of the polymer relative to astation, the identity of the individual unit being unknown, detecting asignal arising from a detectable physical change in the unit or thestation, and distinguishing said signal from signals arising fromexposure of adjacent signal generating units of the polymer to thestation as an indication of the identity of the individual unit. Thisaspect of the invention also encompasses each of the embodimentsdiscussed above.

In one embodiment the station is an interaction station and theindividual units are exposed at the interaction station to an agent thatinteracts with the individual unit to produce a detectableelectromagnetic radiation signal characteristic of the interaction. Inanother embodiment the station is a signal generation station and thecharacteristic signal produced is a polymer dependent impulse.

In yet another aspect the invention is a method for determining theproximity of two individual units of a polymer of linked units. Themethod includes the steps of moving the polymer relative to a station,exposing individual units to the station to produce a characteristicsignal arising from a detectable physical change in the unit or thestation, detecting characteristic signals generated, and measuring theamount of time elapsed between detecting characteristic signals, theamount of time elapsed being indicative of the proximity of the twoindividual units.

In one embodiment the station is an interaction station. In anotherembodiment the interaction station includes an agent and the agent isselected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source and thecharacteristic signal is a detectable electromagnetic radiation signal.In another embodiment the interaction station is a nanochannel in a wallmaterial.

In certain other embodiments the station referred to is a signalgeneration station. In another embodiment the signal generation stationincludes a physical impulse source which interacts with the polymer toproduce a characteristic signal which is a physical impulse. Thephysical impulse in one embodiment arises from a change in a physicalquantity such as resistance or conductance as a result of the exposureof the physical impulse source to the unit of the polymer. In oneembodiment the physical impulse arises from changes in capacitance orresistance caused by the movement of the unit between microelectrodes ornanoelectrodes positioned adjacent to the polymer unit. For instance thesignal generation station may include microelectrodes or nanoelectrodespositioned on opposite sides of the polymer unit. The changes inresistance or conductance which occur as a result of the movement of theunit past the electrodes will be specific for the particular unit. Inanother embodiment the physical impulse arises from a release ofradioactive signal from the unit. In other embodiments it arises frompiezoelectric tip, direct physical contact, and NMR-nuclear spin signal.

The polymer may be any type of polymer known in the art. In a preferredembodiment the polymer is selected from the group consisting of anucleic acid and a protein. In a more preferred embodiment the polymeris a nucleic acid. The polymers can be labeled, randomly or nonrandomly. Different labels can be used to label different linked unitsto produce different signals. In one embodiment the individual units ofthe polymer are labeled with a fluorophore.

A method for determining the order of two individual units of a polymerof linked units is provided in another aspect of the invention. Themethod involves the steps of moving the polymer linearly with respect toa station, exposing one of the individual units to the station toproduce a signal arising from a detectable physical change in the unitor the station, exposing the other of the individual units to thestation to produce a second detectable signal arising from a detectablephysical change in the unit or the station, different from the firstsignal, and determining the order of the signals as an indication of theorder of the two individual units.

In one embodiment the station is an interaction station. In anotherembodiment the interaction station includes an agent and the agent isselected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source and thecharacteristic signals produced are detectable electromagnetic radiationsignals. In another embodiment the interaction station is a nanochannelin a wall material.

In certain other embodiments the station referred to is a signalgeneration station. In another embodiment the signal generation stationincludes a physical impulse source which interacts with the polymer toproduce a characteristic signal which is a physical impulse. Thephysical impulse in one embodiment arises from a change in a physicalquantity such as resistance or conductance as a result of the exposureof the physical impulse source to the unit of the polymer. In oneembodiment the physical impulse arises from changes in capacitance orresistance caused by the movement of the unit between microelectrodes ornanoelectrodes positioned adjacent to the polymer unit. For instance thesignal generation station may include microelectrodes or nanoelectrodespositioned on opposite sides of the polymer unit. The changes inresistance or conductance which occur as a result of the movement of theunit past the electrodes will be specific for the particular unit. Inanother embodiment the physical impulse arises from a release ofradioactive signal from the unit. In other embodiments it arises frompiezoelectric tip, direct physical contact, and NMR-nuclear spin signal.

The polymer may be any type of polymer known in the art. In a preferredembodiment the polymer is selected from the group consisting of anucleic acid and a protein. In a more preferred embodiment the polymeris a nucleic acid. The polymers can be labeled, randomly or nonrandomly. Different labels can be used to label different linked unitsto produce different signals. In one embodiment the individual units ofthe polymer are labeled with a fluorophore. In another embodiment theindividual units of the polymer are labeled with radioactivity.

According to yet another aspect of the invention a method fordetermining the distance between two individual units of a polymer oflinked units is provided. The method involves the steps of causing thepolymer to pass linearly relative to a station, detecting acharacteristic signal generated as each of the two individual unitspasses by the station, measuring the time elapsed between the signalsmeasured, repeating steps 1, 2 and 3 for a plurality of similar polymersto produce a data set, and determining the distance between the twoindividual units based upon the information obtained from said pluralityof similar polymers by analyzing the data set.

In one embodiment the station is an interaction station. In anotherembodiment the interaction station includes an agent and the agent isselected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source. In anotherembodiment the characteristic signals produced are detectableelectromagnetic radiation signals. In another embodiment the interactionstation is a nanochannel in a wall material.

In certain other embodiments the station referred to is a signalgeneration station. In another embodiment the signal generation stationincludes a physical impulse source which interacts with the polymer toproduce a characteristic signal which is a physical impulse. Thephysical impulse in one embodiment arises from a change in a physicalquantity such as resistance or conductance as a result of the exposureof the physical impulse source to the unit of the polymer. In oneembodiment the physical impulse arises from changes in capacitance orresistance caused by the movement of the unit between microelectrodes ornanoelectrodes positioned adjacent to the polymer unit. For instance thesignal generation station may include microelectrodes or nanoelectrodespositioned on opposite sides of the polymer unit. The changes inresistance or conductance which occur as a result of the movement of theunit past the electrodes will be specific for the particular unit. Inanother embodiment the two linked units are detected at the signalgeneration station by measuring light emission at the station. Inanother embodiment the physical impulse arises from a release ofradioactive signal from the unit. In other embodiments it arises frompiezoelectric tip, direct physical contact, and NMR-nuclear spin signal.

The polymer may be any type of polymer known in the art. In a preferredembodiment the polymer is selected from the group consisting of anucleic acid and a protein. In a more preferred embodiment the polymeris a nucleic acid. The polymers can be labeled, randomly or nonrandomly. Different labels can be used to label different linked unitsto produce different signals. In one embodiment the individual units ofthe polymer are labeled with a fluorophore.

According to another embodiment the plurality of similar polymers is ahomogeneous population. In another embodiment the plurality of similarpolymers is a heterogenous population.

In another embodiment steps (1)-(4) are carried out substantiallysimultaneously.

According to yet another aspect of the invention a method for detectingresonance energy transfer or quenching between two interactive partnerscapable of such transfer or quenching is disclosed. The method involvesthe steps of bringing the two partners in close enough proximity topermit such transfer or quenching, applying an agent to one of saidpartners, the agent selected from the group consisting ofelectromagnetic radiation, a quenching source and a fluorescenceexcitation source, shielding fluorescence resonance energy transfer andquenching occurring from electromagnetic radiation emission andinteraction between said partners with a material shield, and detectingthe emitted electromagnetic radiation. In a preferred embodiment thematerial shield is a conductive material shield.

In another aspect the invention is a method for analyzing a polymer oflinked units. The method includes the steps of providing a labeledpolymer of linked units, detecting signals from unit specific markers ofless than all of the linked units, and storing a signature of saidsignals detected to analyze the polymer. In one embodiment all of theunit specific markers are detected. In another embodiment the polymer ispartially and randomly labeled with unit specific markers. In yetanother embodiment only a portion of the unit specific markers aredetected. All of the units of the polymer are labeled with a unitspecific marker in another embodiment.

The labeled polymer of linked units in one embodiment is exposed to anagent selected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source and the signalsare produced by the interaction between a unit specific marker of thepolymer and the agent.

In one embodiment the signals are detected linearly. In anotherembodiment the signature of signals includes at least 10 signals, andpreferably 20 signals. The signature of signals includes any informationabout the polymer. Preferably the signature of signals includesinformation about the order, distance and number of unit specificmarkers.

In another embodiment the labeled polymer of linked units is moved withrespect to a station and wherein the signals are generated upon exposureof a unit specific marker of the polymer to the station. The station maybe an interaction station.

The method in some embodiments is a method for identifying a unitspecific marker of the polymer, the identity of the unit specific markerbeing indicative of the identity of at least one unit of the polymer.The unit specific marker is transiently exposed to a station to producesignals characteristic of said unit specific marker and the signal isdistinguished from signals generated from adjacent signal generatingunit specific markers of the polymer as an indication of the identity ofthe unit specific marker. The station may be an interaction stationincluding an agent selected from the group consisting of electromagneticradiation, a quenching source and a fluorescence excitation source andwherein the signals are detectable electromagnetic radiation signals.

The method in other embodiments is a method for determining theproximity of two unit specific markers of the polymer wherein theproximity of the two unit specific markers is the signature of saidsignals, the identity of each unit specific marker being indicative ofthe identity of at least one unit of the polymer. The labeled polymer ismoved relative to a station to expose the two unit specific markers tothe station to produce a characteristic signal arising from a detectablephysical change in the unit specific marker or the station, and theamount of time elapsed between detecting each characteristic signal ismeasured, the amount of time elapsed being indicative of the proximityof the two unit specific markers.

The method may also be a method for determining the order of two unitspecific markers of the polymer, the identity of each unit specificmarker being indicative of the identity of at least one unit of thepolymer wherein the order of the two unit specific markers is thesignature of said signals. The labeled polymer is moved linearly withrespect to a station, to expose one of the unit specific markers to thestation to produce a signal which is a unit specific marker and toexpose the other of the unit specific markers to the station to producea second detectable which is a unit specific marker, different from thefirst signal. The order of the signals determined is an indication ofthe order of the two unit specific markers.

The method in an embodiment is a method for determining the distancebetween two unit specific markers of the polymer, the identity of eachunit specific marker being indicative of the identity of at least oneunit of the polymer wherein the distance between two unit specificmarkers is the signature of said signals. The labeled polymer is movedlinearly relative to a station to produce a characteristic signalgenerated as each of the two unit specific markers passes by the stationand the distance between the signals is determined as an indication ofthe distance between the two unit specific markers.

The method is a method for characterizing a test labeled polymer,wherein a plurality of labeled polymers is exposed to a station toobtain the signature of signals for each of the plurality of labeledpolymers in another embodiment. The method involves the steps ofcomparing the signature of signals of the plurality of polymers,determining the relatedness of the polymers based upon similaritiesbetween the signature of signals of the polymers, and characterizing thetest polymer based upon the signature of signals of related polymers.

According to yet another embodiment the method is a method forsequencing a polymer of linked units. A signature of signals is obtainedfrom each of a plurality of overlapping polymers, at least a portion ofeach of the polymers having a sequence of linked units identical to theother of the polymers, and the signature of signals is compared toobtain a sequence of linked units which is identical in the plurality ofpolymers.

The method in another embodiment is a method for analyzing a set ofpolymers, each polymer of said set being an individual polymer of linkedunits and wherein the set of polymers is oriented parallel to oneanother and a polymer specific feature of said polymers is detected.

Each of the above methods is based on an interaction between a polymerand a station involving in some embodiments energy transfer or quenchingbetween a unit and an agent which results in the generation of a signaland in other embodiments a physical change in the unit or station whichresults in the generation of a signal. Each of the methods can beperformed on many polymers simultaneously or on as few as one polymer ata time.

Methods for analyzing multiple polymers at one time based on aninteraction involving polymer dependent impulses between the unit andthe station also can be performed. These methods, which are set forthbelow, are based on an interaction between a unit and a signalgeneration station which produces any type of polymer dependent impulsewhich can be detected. The polymer dependent impulse is generated byexposure of a unit of the polymer to a signal generation station butdoes not require that a physical change in the polymer unit or thestation occur. For instance, the polymer dependent impulse may resultfrom energy transfer, quenching, changes in conductance, mechanicalchanges, resistance changes, or any other physical change.

A method for characterizing a test polymer is another aspect of theinvention. A method for characterizing a test polymer is carried out byobtaining polymer dependent impulses for each of a plurality ofpolymers, comparing the polymer dependent impulses of the plurality ofpolymers, determining the relatedness of the polymers based uponsimilarities between the polymer dependent impulses of the polymers, andcharacterizing the test polymer based upon the polymer dependentimpulses of related polymers.

The plurality of polymers may be any type of polymer but preferably is anucleic acid. In one embodiment the plurality of polymers is ahomogenous population. In another embodiment the plurality of polymersis a heterogenous population. The polymers can be labeled, randomly ornon randomly. Different labels can be used to label different linkedunits to produce different polymer dependent impulses.

The polymer dependent impulses provide many different types ofstructural information about the polymer. For instance the obtainedpolymer dependent impulses may include an order of polymer dependentimpulses or the obtained polymer dependent impulses may include the timeof separation between specific signals or the number of specific polymerdependent impulses.

In one important embodiment the polymer dependent impulses are obtainedby moving the plurality of polymers linearly past a signal generationstation.

According to another aspect the invention is a method for determiningthe distance between two individual units of a polymer of linked units.The method involves the steps of (1) causing the polymer to passlinearly relative to a station, (2) detecting a polymer dependentimpulse generated as each of the two individual units passes by thesignal generation station, (3) measuring the time elapsed between thepolymer dependent impulses measured, (4) repeating steps 1, 2 and 3 fora plurality of similar polymers to produce a data set, and (5)determining the distance between the two individual units based upon theinformation obtained from said plurality of similar polymers byanalyzing the data set. In one embodiment steps (1)-(4) are carried outsubstantially simultaneously.

The plurality of polymers may be any type of polymer but preferably is anucleic acid. In one embodiment the plurality of polymers is ahomogenous population. In another embodiment the plurality of polymersis a heterogenous population. The polymers can be labeled, randomly ornon randomly. Different labels can be used to label different linkedunits to produce different polymer dependent impulses. In one embodimentthe polymer dependent impulse measured is an electromagnetic radiationsignal generated. In another embodiment the two linked units aredetected at the signal generation station by measuring light emission atthe station. The signal generation station can be a nanochannel.

According to another aspect the invention is a method for determiningthe order of two individual units of a polymer of linked units. Themethod involves the steps of (1) moving the polymer to linearly withrespect to a signal generation station, (2) exposing one of theindividual units to the station to produce a polymer dependent impulse,(3) exposing the other of the individual units to the station to producea second polymer dependent impulse, (4) repeating steps 1, 2 and 3 for aplurality of similar polymers to produce a data set, and (5) determiningthe order of the two individual units based upon the informationobtained from said plurality of similar polymers by analyzing the dataset. In one embodiment steps (1)-(4) are carried out substantiallysimultaneously. In one embodiment the signal measured is anelectromagnetic radiation signal.

The plurality of polymers may be any type of polymer but preferably is anucleic acid. In one embodiment the plurality of polymers is ahomogenous population. In another embodiment the plurality of polymersis a heterogenous population. The polymers can be labeled, randomly ornon randomly. Different labels can be used to label different linkedunits to produce different polymer dependent impulses.

In one embodiment the polymer dependent impulse measured is anelectromagnetic radiation signal generated. In another embodiment thetwo linked units are detected at the signal generation station bymeasuring light emission at the station. The signal generation stationcan be a nanochannel.

In another aspect of the invention a method for sequencing a polymer oflinked units is provided. The method involves the steps of obtainingpolymer dependent impulses from a plurality of overlapping polymers, atleast a portion of the polymers having a sequence of linked unitsidentical to the other of the polymers, and comparing the polymerdependent impulses to obtain a sequence of linked units which isidentical in the plurality of polymers.

In one embodiment the polymer dependent impulses are opticallydetectable. In another embodiment the nucleic acids are labeled with anagent selected from the group consisting of an electromagnetic radiationsource, a quenching source, a fluorescence excitation source, and aradiation source.

The plurality of polymers may be any type of polymer but preferably is anucleic acid. In one embodiment the plurality of polymers is ahomogenous population. In another embodiment the plurality of polymersis a heterogenous population. The polymers can be labeled, randomly ornon randomly. Different labels can be used to label different linkedunits to produce different polymer dependent impulses.

A method for labeling nucleic acids is also provided. The methodinvolves the step of contacting a dividing cell with a nucleotideanalog, isolating from the cell nucleic acids that have incorporated thenucleotide analog, and modifying the nucleic acid with incorporatednucleotide analog by labeling the incorporated nucleotide analog. In oneembodiment the nucleotide analog is a brominated analog.

The dividing cell may optionally be contacted with a nucleotide analogby growth arresting the cell in the cell division cycle, performing thecontacting step, and allowing the cell to reenter the cell divisioncycle. The nucleic acids may then be isolated after the cells havereentered and completed the cell division cycle and before a second celldivision cycle is completed.

In another embodiment the incorporated nucleotide analog is labeled withan agent selected from the group consisting of an electromagneticradiation source, a quenching source and a fluorescence excitationsource.

According to another aspect of the invention a method is provided foranalyzing a set of polymers, each polymer of said set being anindividual polymer of linked units. The method involves the step oforienting the set of polymers parallel to one another, and detecting apolymer specific feature of said polymers. In one embodiment theorientation step is in a solution free of gel. The polymers may beoriented using any method. A preferred method for orienting the polymersis to apply an electric field to the polymers.

The plurality of polymers may be any type of polymer but preferably is anucleic acid. In one embodiment the plurality of polymers is ahomogenous population. In another embodiment the plurality of polymersis a heterogenous population. The polymers can be labeled, randomly ornon randomly. Different labels can be used to label different linkedunits to produce different polymer dependent impulses.

The polymer specific feature is information about a structural featureof a polymer. The polymer specific feature can be an order of linkedunity in the polymers.

In one embodiment the detecting step is performed simultaneously forsaid polymers. In another embodiment the detection step comprisesmeasuring electromagnetic radiation signals. According to a preferredembodiment the detection step comprises causing the polymers to passlinearly relative to a plurality of signal generation stations, anddetecting and distinguishing polymer dependent impulses generated assaid polymers pass said signal generation stations.

A method for analyzing a set of polymers, each polymer of the set beingan individual polymer of linked units is provided in another aspect ofthe invention. The method involves the steps of orienting the set ofpolymers in an electric field, simultaneously moving the set of polymersthrough defined respective channels, and detecting a polymer specificfeature as the polymers are moved through the channels. In oneembodiment the orientation step is in a solution free of gel. Preferablythe channels are nanochannels.

The plurality of polymers may be any type of polymer but preferably is anucleic acid. In one embodiment the plurality of polymers is ahomogenous population. In another embodiment the plurality of polymersis a heterogenous population. The polymers can be labeled, randomly ornon randomly. Different labels can be used to label different linkedunits to produce different polymer dependent impulses.

The polymer specific feature is information about a structural featureof a polymer. The polymer specific feature can be an order of linkedunity in the polymers.

In one embodiment the detecting step is performed simultaneously forsaid polymers. In another embodiment the detection step comprisesmeasuring electromagnetic radiation signals. According to a preferredembodiment the detection step comprises causing the polymers to passlinearly relative to a plurality of signal generation stations, anddetecting and distinguishing polymer dependent impulses generated assaid polymers pass said signal generation stations.

According to yet another aspect of the invention an article ofmanufacture is provided. The article of manufacture includes a wallmaterial having a surface defining a channel, an agent wherein the agentis selected from the group consisting of an electromagnetic radiationsource, a quenching source, a luminescent film layer and a fluorescenceexcitation source, attached to the wall material adjacent to thechannel, wherein the agent is close enough to the channel and is presentin an amount sufficient to detectably interact with a partner compoundselected from the group consisting of a light emissive compound, a lightaccepting compound, radiative compound, and a quencher passing throughthe channel. Preferably the channel is a support for a polymer.

The agent in one embodiment is an electromagnetic radiation source andthe electromagnetic radiation source is a light emissive compound. Inanother embodiment the channel is selected from the group consisting ofis a microchannel and a nanochannel.

According to another embodiment the surface of the wall materialdefining the channel is free of the light emissive compound. In anotherembodiment the light emissive compound is attached to an externalsurface of the wall material. In yet another embodiment the lightemissive compound is attached to a linker which is attached to theexternal surface of the wall material. In still another embodiment thelight emissive compound is embedded in the wall material or in a layerof or upon the wall material. The light emissive compound can beconcentrated at a region of the external surface of the wall materialthat surrounds a portion of the channel in another embodiment. The lightemissive compound may form a concentric ring in the wall material arounda portion of the channel. A masking layer having openings which allowexposure of only localized areas of the light emissive compound may alsobe part of the article of manufacture.

A second light emissive compound different from the first may beattached to the wall material adjacent to the channel, wherein the lightemissive compound is close enough to the channel and is present in anamount effective to detectably interact with a partner light emissivecompound passing through the channel.

The wall material may be made up of different layers. In one embodimentthe external surface of the wall material adjacent to the light emissivecompound is a conducting layer. In another embodiment the wall materialcomprises two layers, the conducting layer and a nonconducting layer.The wall material may also be composed of at least two layers, a firstlayer preventing signal generation and a second layer allowing signalgeneration. Alternatively the wall material adjacent to the lightemissive compound is a light impermeable layer. In another embodimentthe wall material comprises two layers, the light impermeable layer anda support light permeable layer. The wall material can be a second lightimpermeable layer on a second side of the light emissive compound, thefirst and second layers sandwiching the light emissive compound. In apreferred embodiment the light emissive compound is a fluorescentcompound.

The channel can have any shape or dimensions. Preferably the channel isa nanochannel which is between 1 Angstrom and 1 mm. In a preferredembodiment the width of the channel is between 1 and 500 Angstroms.Preferably the wall includes multiple channels. Preferably the wallmaterial includes at least 2 and more preferably at least 50 channels.

In one embodiment the wall material is formed of two layers, a firstlight impermeable layer and a luminescent film layer attached to oneanother, wherein the channel extends through both layers and is definedby surfaces of both layers. Preferably the channel is a nanochannel. Ina preferred embodiment the length of the channel is between 1 Angstromand 1 mm. The article in some embodiments includes a second lightimpermeable layer, the luminescent film layer positioned between thefirst and second light impermeable layers. In a preferred embodiment thesurface defining the channel includes a surface of the light impermeablelayer which is free of luminescent film layer material.

In another embodiment the agent is a fluorescence excitation source andwherein the fluorescence excitation source is a scintillation layer. Thescintillation layer may be selected from the group consisting ofNaI(TI), ZnS(Ag), anthracene, stilbene, and plastic phosphors.Preferably the scintillation layer is embedded in the wall materialbetween two radiation impermeable layers, such as lead or Lucite.

In another aspect the invention is an article of manufacture which is awall material having a surface defining a plurality of channels, and astation attached to a discrete region of the wall material adjacent toat least one of the channels, wherein the station is close enough to thechannel and is present in an amount sufficient to cause a signal toarise from a detectable physical change in a polymer of linked unitspassing through the channel or in the station as the polymer is exposedto the station.

According to another aspect of the invention an article of manufactureis provided. The article is a wall material having a surface defining achannel, and a plurality of stations each attached to a discrete regionof the wall material adjacent to the channel, wherein the stations areclose enough to the channel and are present in an amount sufficient tocause a signal to arise from a detectable physical change in a polymerof linked units passing through the channel or in the station as thepolymer is exposed to the station.

A method for preparing a wall material is another aspect of theinvention. The method involves the steps of covalently bonding lightemissive compounds or quenching compounds to a plurality of discretelocations of a wall material, each of said discrete locations closeenough to a respective interaction station on said wall material,whereby when an individual unit of a polymer, which is interactive withsaid light emissive compound or quenching compound to produce a signal,is positioned at said interaction station, the light emissive compoundor the quenching compound interacts with the individual unit to producethe signal. In one embodiment the method includes the step of applying alayer of conductive material to said wall material.

In another embodiment the light emissive compounds or quenchingcompounds are covalently bonded at discrete locations close to channelsin said wall material, said channels defining interaction stations. Thechannels preferably are microchannels. In a more preferred embodimentthe channels are nanochannels. The light emissive compounds or quenchingcompounds can be covalently bonded to the wall material in a mannerwhereby the surfaces of the wall material defining the channel are freeof the light emissive compounds and quenching compounds.

The invention also encompasses a method for attaching a chemicalsubstance selectively at a rim of a channel through a wall material thatis opaque. The method involves the steps of providing a wall materialwith photoprotective chemical groups attached at the rim of the channelthrough the wall material, applying light to the photoprotectivechemical groups to dephotoprotect the chemical groups, and attaching thechemical substance to the deprotected chemical groups.

In one embodiment the light is applied to only selected regions of asurface of the wall material defining the rim of the channel. In anotherembodiment the channel has a first end and a second end, the rim beingat the first end, and wherein the light is applied to the second end,the light passing through the channel to contact the photoprotectedchemical groups at the rim of said first end. The channels preferablyare microchannels. In a more preferred embodiment the channels arenanochannels.

According to another aspect of the invention a method is provided forpreparing a wall material having localized areas of light emission on asurface of the wall material. The method involves the steps of providinga wall material having a surface and applying a light emissive compoundto the surface to produce at least localized areas of light emission onthe surface, wherein the localized areas define a target region fordetecting light emission, and wherein the target region is a rim of achannel through the wall material. In one embodiment the method furtherincludes the steps of attaching a photoprotective chemical group to thesurface of the wall material, applying light to the photoprotectivechemical groups to dephotoprotect the chemical groups prior to attachingthe light emissive compound, and attaching the light emissive compoundto the dephotoprotected chemical groups.

In one embodiment the light is applied to only selected regions of asurface of the wall material defining the rim of the channel. In apreferred embodiment the photoprotective chemical group is attached toonly selected regions of the surface of the wall material defining therim of the channel. In another embodiment the channel has a first endand a second end, the rim being at the first end, and wherein the lightis applied to the second end, the light passing through the channel tocontact the photoprotected chemical groups at the rim of said first end.The channels preferably are microchannels. In a more preferredembodiment the channels are nanochannels.

The method can include the further step of positioning a mask havingopenings over the surface of the wall material such that only localizedareas of light emission are exposed through the openings of the mask. Inone embodiment the light emissive compound is attached to a portion ofthe surface of the wall material.

According to another aspect of the invention an apparatus for detectinga signal is provided. The apparatus is a housing with a buffer chamber,a wall defining a portion of the buffer chamber, and having a pluralityof openings for aligning polymers, a sensor fixed relative to thehousing, the sensor distinguishing the signals emitted at each openingfrom the signals emitted at the other of the openings to generateopening dependent sensor signals, and a memory for collecting andstoring said sensor signals. In a preferred embodiment the sensor is anoptical sensor.

In one embodiment the optical sensor senses electromagnetic radiationsignals emitted at the plurality of openings. In another embodiment theapparatus includes a microprocessor.

In one embodiment the openings are defined by channels in the wall.Preferably the openings are defined by microchannels in the wall. Morepreferably the openings are defined by nanochannels in the wall. In oneembodiment the plurality of openings is at least two. In a preferredembodiment the plurality is at least 50.

In one embodiment the apparatus includes a second buffer chamberseparated from said first buffer chamber, by said wall, and wherein thebuffer chambers are in fluid communications with one another via theopenings. In another embodiment the apparatus includes a pair ofelectrodes secured to the housing, one of said pair positioned in thefirst buffer chamber and the other of the pair positioned in the secondbuffer chamber.

According to another aspect of the invention an apparatus for detectinga signal is provided. The apparatus includes a housing defining a firstbuffer chamber and a second buffer chamber, a wall supported by thehousing and separating the first and second buffer chambers, a pluralityof channels defined by the wall and providing fluid communicationsbetween the first and second buffer chambers, and a sensor fordistinguishing and collecting channel dependent signals. Preferably thechannel is a microchannel. More preferably the channel is a nanochannel.In one embodiment the plurality of channels is at least two. In apreferred embodiment the plurality is at least 50. Preferably the signalis an optical signal.

In one embodiment the wall surrounding the channel includes an agent isselected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source is attached to thewall. Preferably the agent is electromagnetic radiation and theelectromagnetic radiation is a light emissive compound. In oneembodiment the light emissive compound is concentrated at the channelsin the wall.

According to another embodiment the apparatus includes a means formoving biological entities through the channels.

According to another aspect of the invention an apparatus including ahousing with a buffer chamber, a wall material defining a portion of thebuffer chamber, the wall including polymer interaction stations, and anoptical sensor secured to the housing, the optical sensor constructedand arranged to detect electromagnetic radiation signals emitted at theinteraction stations is provided.

In another aspect the invention is a computer system for makingcharacteristic information of a plurality of polymers available inresponse to a request. The system has a memory for storing, for theplurality of the polymers and in a manner accessible using a uniqueidentifier for the polymer, records including information indicative ofsequentially detected signals arising from a detectable physical changein the plurality of individual units of the polymer or a station towhich the polymer is exposed and a processor for accessing the recordsstored in the memory for a selected one of the plurality of the polymersaccording to a unique identifier associated with the selected polymer.

In one embodiment the signal results from an interaction of a pluralityof individual units of the polymer exposed to an agent selected from thegroup consisting of electromagnetic radiation, a quenching source and afluorescence excitation source. In another embodiment the computersystem also includes a means for comparing the sequentially detectedsignals of the selected polymer to a known pattern of signalscharacteristic of a known polymer to determine relatedness of theselected polymer to the known polymer.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each apparatus and eachmethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of a random labeling method.

FIG. 2 is a graph of raw data demonstrating changes in energy emissionpatterns to determine distance information through the instantaneousrate method. The changes in energy patterns result from sequentialdetectable signals which when plotted produce a curve that from left toright shows two energy intensity decreases, followed by two energyintensity increases. The rate is 6.8 Å/s and t₁ is the time between theentry of the first and second labels.

FIG. 3 shows a representative population of random A-labeled fragmentsfor a 16-mer with the sequence 5′ ACGTACGTACGTACGT 3′ (SEQ. ID. No. 1)and also depicts how distance information is used to determine baseseparation between acceptor labeled nucleotides.

FIG. 4A-4R is a schematic depicting various potential constructions of ananochannel plate.

FIG. 5 is a schematic depicting a nanochannel plate for analyzing aradioactive polymer.

FIG. 6 is a schematic of a cross section of a nanochannel with aconcentric ring of donor fluorophores and a graph showing donor emissionand energy transfer on the corresponding nanochannel.

FIG. 7A shows a model of a nanochannel plate having multiplenanochannels with a layer of donor fluorophores within the plate. FIG.7B shows an enlarged view of a single nanochannel with a single acceptorpositioned adjacent to a conducting layer.

FIGS. 8A and B are schematics demonstrating signal generation uponpassage of an acceptor label through the nanochannel. FIG. 8A shows anenlarged view of one nanochannel. FIG. 8B graphically illustrates thesharp changes in donor intensity as an acceptor label moves frompositions A to C.

FIG. 9 schematically and graphically demonstrates the passage of atwo-base labeled strand of DNA through a nanochannel with the properthickness and radii of donor fluorophores sandwiched between conductingmaterial.

FIGS. 10A and B are volume graphs visually demonstrating the change indonor emission in the presence of one and two acceptors. FIG. 10Aillustrates the amount of change as volumes. FIG. 10B shows the changefor one acceptor.

FIG. 11 shows the results of an experiment demonstrating that DNA canpass through fabricated nanochannels.

FIG. 12 shows an apparatus constructed to hold a nanochannel (ormicrochannel) plate which is capable of generating an electric field.

FIG. 13 depicts a nanochannel apparatus which consists of two fusedPyrex cells that hold the nanochannel plate, an upper and lower bufferregion, electrodes, an immersion objective and a voltage supply.

FIG. 14 is a schematic diagram of in vitro base specific (IBSA)labeling.

FIG. 15 shows the general scheme for deciphering DNA sequence from IBSAlabeling.

FIG. 16 includes a graph illustrating the relationship of emissions todistance (Angstroms).

DETAILED DESCRIPTION OF THE INVENTION

The invention encompasses methods of analyzing or identifying a polymeror a unit of a polymer, by detecting a signal or polymer dependentimpulse that results from an interaction between at least one unit ofthe polymer and a station or an agent or by a change in the unit or astation when the unit is exposed to the station. By “analyzing” apolymer, it is meant obtaining some information about the structure ofthe polymer such as its size, the order of its units, its relatedness toother polymers, the identity of its units, or its presence. Since thestructure and function of biological molecules are interdependent, thestructural information can reveal important information about thefunction of the polymer.

One method according to the invention is a method for analyzing apolymer of linked units by exposing a plurality of units of the polymerto an agent selected from the group consisting of electromagneticradiation, a quenching source and a fluorescence excitation source suchthat each of the individual units interacts with the agent to produce adetectable signal. The signal resulting from the interaction is detectedsequentially.

As used herein a unit of a polymer is “exposed” to an agent or a stationby positioning or presenting the unit and the agent or station ininteractive proximity to one another such that energy transfer orquenching or a physical change in the unit or agent or station can occurbetween them to produce a detectable signal. By interactive proximity itis meant close enough to permit the interaction or change which yieldsthe detectable signal.

In one embodiment the units of the polymer are exposed sequentially tothe agent. By “sequentially exposed” it is meant in general separatedfrom one another in time. In a preferred embodiment, the polymer and theagent are caused to move relative to one another in a “linear” mannersuch that each unit of the polymer passes within interactive proximityto the agent at an interaction station. When each unit of the polymerinteracts with the agent or station, a detectable signal is produced.This detectable signal can be captured (sequentially detected) andrecorded by a detection device. The detectable signal produced for eachunit can be indicative of the type of unit. As used herein signals aredetected “sequentially” when signals from different units of a singlepolymer are detected spaced apart in time. Not all units need to bedetected or need to generate a signal to detect signals “sequentially.”

When the units are sequentially exposed to the agent or station the unitand the agent or station move relative to one another. As used hereinthe phrase “the unit and the agent or station move relative to oneanother” means that either the unit and the agent or station are bothmoving or only one of the two is moving and the other remains stationaryat least during the period of time of the interaction between the unitand the agent or station. The unit and the agent or station may be movedrelative to one another by any mechanism. For instance the agent orstation may remain stationary and the polymer may be drawn past theagent or station by an electric current. Other methods for moving thepolymer include but are not limited to movement resulting from amagnetic field, a mechanical force, a flowing liquid medium, a pressuresystem, a gravitational force, and a molecular motor such as e.g., a DNApolymerase or a helicase when the polymer is DNA or e.g., myosin whenthe polymer is a peptide such as actin. The movement of the polymer maybe assisted by the use of a channel, groove or ring to guide thepolymer. Alternatively the agent or station may be moved and the polymermay remain stationary. For instance the agent or station may be heldwithin a scanning tip that is guided along the length of the polymer.

In another embodiment signals are detected simultaneously. As usedherein signals are “detected simultaneously” by causing a plurality ofthe labeled units of a polymer to be exposed to an agent or station atonce. The plurality of the units can be exposed to an agent or stationat one time by using multiple interaction sites. Signals can be detectedat each of these sites simultaneously. For instance multiple agents orstations may be localized at specific locations in space whichcorrespond to the units of the polymer. When the polymer is broughtwithin interactive proximity of the multiple agents or stations signalswill be generated simultaneously. This may be embodied, for example, ina linear array of agents or stations positioned at substantiallyequivalent distances which are equal to the distance between the units.The polymer may be positioned with respect to the agent or station suchthat each unit is in interactive proximity to an agent or station toproduce simultaneous signals.

When the signals are detected sequentially multiple polymers also can beanalyzed simultaneously. Multiple polymers are analyzed simultaneouslyby causing more than one polymer to move relative to respective agent orstations at one time. The polymers may be similar or distinct. If thepolymers are similar, the same or different units may be detectedsimultaneously. It is preferred that at least two polymers are analyzedsimultaneously. In a more preferred embodiment at least 50 polymers areanalyzed simultaneously and in another preferred embodiment at least 100polymers are analyzed simultaneously.

A “polymer” as used herein is a compound having a linear backbone ofindividual units which are linked together by linkages. In some casesthe backbone of the polymer may be branched. Preferably the backbone isunbranched. The term “backbone” is given its usual meaning in the fieldof polymer chemistry. The polymers may be heterogeneous in backbonecomposition thereby containing any possible combination of polymer unitslinked together such as peptide-nucleic acids (which have amino acidslinked to nucleic acids and have enhanced stability). In a preferredembodiment the polymers are homogeneous in backbone composition and are,for example, nucleic acids, polypeptides, polysaccharides,carbohydrates, polyurethanes, polycarbonates, polyureas,polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides,polyacetates, polyamides, polyesters, or polythioesters. In the mostpreferred embodiments, the polymer is a nucleic acid or a polypeptide. A“nucleic acid” as used herein is a biopolymer comprised of nucleotides,such as deoxyribose nucleic acid (DNA) or ribose nucleic acid (RNA). Apolypeptide as used herein is a biopolymer comprised of linked aminoacids.

As used herein with respect to linked units of a polymer, “linked” or“linkage” means two entities are bound to one another by anyphysicochemical means. Any linkage known to those of ordinary skill inthe art, covalent or non-covalent, is embraced. Such linkages are wellknown to those of ordinary skill in the art. Natural linkages, which arethose ordinarily found in nature connecting the individual units of aparticular polymer, are most common. Natural linkages include, forinstance, amide, ester and thioester linkages. The individual units of apolymer analyzed by the methods of the invention may be linked, however,by synthetic or modified linkages. Polymers where the units are linkedby covalent bonds will be most common but also include hydrogen bonded,etc.

The polymer is made up of a plurality of individual units. An“individual unit” as used herein is a building block or monomer whichcan be linked directly or indirectly to other building blocks ormonomers to form a polymer. The polymer preferably is a polymer of atleast two different linked units. The at least two different linkedunits may produce or be labeled to produce different signals, asdiscussed in greater detail below. The particular type of unit willdepend on the type of polymer. For instance DNA is a biopolymercomprised of a deoxyribose phosphate backbone composed of units ofpurines and pyrimidines such as adenine, cytosine, guanine, thymine,5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,2,6-diaminopurine, hypoxanthine, and other naturally and non-naturallyoccurring nucleobases, substituted and unsubstituted aromatic moieties.RNA is a biopolymer comprised of a ribose phosphate backbone composed ofunits of purines and pyrimidines such as those described for DNA butwherein uracil is substituted for thymidine. DNA units may be linked tothe other units of the polymer by their 5′ or 3′ hydroxyl group therebyforming an ester linkage. RNA units may be linked to the other units ofthe polymer by their 5′, 3′ or 2′ hydroxyl group thereby forming anester linkage. Alternatively, DNA or RNA units having a terminal 5′, 3′or 2′ amino group may be linked to the other units of the polymer by theamino group thereby forming an amide linkage. The individual units of apolypeptide are amino acids, including the 20 naturally occurring aminoacids as well as modified amino acids. Amino acids may exist as amidesor free acids and are linked to the other units in the backbone of thepolymers through their α-amino group thereby forming an amide linkage tothe polymer.

A “plurality of individual units” is at least two units linked to oneanother.

Whenever a nucleic acid is represented by a sequence of letters it willbe understood that the nucleotides are in 5′→3′ order from left to rightand that “A” denotes adenosine, “C” denotes cytidine, “G” denotesguanosine, “T” denotes thymidine, and “U” denotes uracil unlessotherwise noted.

The polymers may be native or naturally-occurring polymers which occurin nature or non-naturally occurring polymers which do not exist innature. The polymers typically include at least a portion of a naturallyoccurring polymer. The polymers can be isolated or synthesized de novo.For example, the polymers can be isolated from natural sources e.g.purified, as by cleavage and gel separation or may be synthesized e.g.,(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) synthesized by, for example, chemical synthesis; (iii)recombinantly produced by cloning, etc.

The polymer or at least one unit thereof is in a form which is capableof interacting with an agent or station to produce a signalcharacteristic of that interaction. The unit of a polymer which iscapable of undergoing such an interaction is said to be labeled. If aunit of a polymer can undergo that interaction to produce acharacteristic signal, then the polymer is said to be intrinsicallylabeled. It is not necessary that an extrinsic label be added to thepolymer. If a non-native molecule, however, must be attached to theindividual unit of the polymer to generate the interaction producing thecharacteristic signal, then the polymer is said to be extrinsicallylabeled. The “label” may be, for example, light emitting, energyaccepting, fluorescent, radioactive, or quenching.

Many naturally occurring units of a polymer are light emitting compoundsor quenchers. For instance, nucleotides of native nucleic acid moleculeshave distinct absorption spectra, e.g., A, G, T, C, and U haveabsorption maximums at 259 nm, 252 nm, 267 nm, 271 nm, and 258 nmrespectively. Modified units which include intrinsic labels may also beincorporated into polymers. A nucleic acid molecule may include, forexample, any of the following modified nucleotide units which have thecharacteristic energy emission patterns of a light emitting compound ora quenching compound: 2,4-dithiouracil, 2,4-Diselenouracil,hypoxanthine, mercaptopurine, 2-aminopurine, and selenopurine.

A unit may also be considered to be intrinsically labeled when aproperty of the unit other than a light emitting, quenching orradioactive property provides information about the identity of the unitwithout the addition of an extrinsic label. For instance the shape andcharge of the unit provides information about the unit which can resultin a specific characteristic signal, such as a change in conductancearising from the blockage of a conductance path by the unit.

If an extrinsic label is selected for use according to the methods ofthe invention, the type of extrinsic label selected will depend on avariety of factors, including the nature of the analysis beingconducted, the type of the agent and the type of polymer. Extrinsiclabel compounds include but are not limited to light emitting compounds,quenching compounds, radioactive compounds, spin labels, and heavy metalcompounds. The label should be stearically compatible and chemicallycompatible with the units of the polymer being analyzed.

A “light emissive compound” as used herein is a compound that emitslight in response to irradiation with light of a particular wavelength.These compounds are capable of absorbing and emitting light throughphosphorescence, chemiluminescence, luminescence, polarizedfluorescence, scintillators or, more preferably, fluorescence. Theparticular light emissive compound selected will depend on a variety offactors which are discussed in greater detail below. Light emissivecompounds have been described extensively in the literature. Forexample, Haugland, R. P., Handbook of Fluorescent Probes and ResearchChemicals, 6th edition, Molecular Probes, Inc., 1996, which is herebyincorporated by reference provides a description of light emittingcompounds.

Radioactive compounds are substances which emit alpha, beta, or gammanuclear radiation. Alpha rays are positively charged particles of massnumber 4 and slightly deflected by electrical and magnetic fields. Betarays are negatively charged electrons and are strongly deflected byelectrical and magnetic fields. Gamma rays are photons ofelectromagnetic radiation and are undeflected by electrical and magneticfields and are of wavelength of the order to 10⁻⁸ to 10⁻⁹ cm.

Generally, fluorescent dyes are hydrocarbon molecules having a chain ofseveral conjugated double bonds. The absorption and emission wavelengthsof a dye are approximately proportional to the number of carbon atoms inthe conjugated chain. A preferred fluorescent compound is “Cy-3”(Biological Detection Systems, Pittsburgh, Pa.). Other preferredfluorescent compounds useful according to the invention include but arenot limited to fluorescein isothiocyanate (“FITC”), Texas red,tetramethylrhodamine isothiocyanate (“TRITC”), 4,4-difluoro-4-bora-3a,and 4a-diaza-s-indacene (“BODIPY”).

Chemiluminescent compounds are compounds which luminesce due to achemical reaction. Phosphorescent compounds are compounds which exhibitdelayed luminescence as a result of the absorption of radiation.Luminescence is a non-thermal emission of electromagnetic radiation by amaterial upon excitation. These compounds are well known in the art andare available from a variety of sources.

In one embodiment of the invention the light emissive compound is adonor or an acceptor fluorophore. A fluorophore as used herein is amolecule capable of absorbing light at one wavelength and emitting lightat another wavelength. A donor fluorophore is a fluorophore which iscapable of transferring its fluorescent energy to an acceptor moleculein close proximity. An acceptor fluorophore is a fluorophore that canaccept energy from a donor at close proximity. (An acceptor of a donorfluorophore does not have to be a fluorophore. It may benon-fluorescent.) Fluorophores can be photochemically promoted to anexcited state, or higher energy level, by irradiating them with light.Excitation wavelengths are generally in the UV, blue, or green regionsof the spectrum. The fluorophores remain in the excited state for a veryshort period of time before releasing their energy and returning to theground state. Those fluorophores that dissipate their energy as emittedlight are donor fluorophores. The wavelength distribution of theoutgoing photons forms the emission spectrum, which peaks at longerwavelengths (lower energies) than the excitation spectrum, but isequally characteristic for a particular fluorophore.

In another embodiment of the invention the unit is labeled with aradioactive compound. The radioactive compound emits nuclear radiationas it passes the agent or station. When the agent is a scintillationlayer the nuclear radiation interacts with the scintillation layer andcauses fluorescent excitation. A fluorescent signal indicative of theradioactively labeled unit can then be detected.

Extrinsic labels can be added to the polymer by any means known in theart. For example, the labels may be attached directly to the polymer orattached to a linker which is attached to the polymer. For instance,fluorophores have been directly incorporated into nucleic acids bychemical means but have also been introduced into nucleic acids throughactive amino or thio groups into a nucleic acid. Proudnikov andMirabekov, Nucleic Acids Research, 24: 4535-4532 (1996). Modified unitswhich can easily be chemically derivitized or which include linkers canbe incorporated into the polymer to enhance this process. An extensivedescription of modification procedures which can be performed on thepolymer, the linker and/or the extrinsic label in order to prepare abioconjugate can be found in Hermanson, G. T., Bioconjugate Techniques,Academic Press, Inc., San Diego, 1996, which is hereby incorporated byreference.

There are several known methods of direct chemical labeling of DNA(Hermanson, 1996; Roget et al., 1989; Proudnikov and Mirzzbekov, 1996).One of the methods is based on the introduction of aldehyde groups bypartial depurination of DNA. Fluorescent labels with an attachedhydrazine group are efficiently coupled with the aldehyde groups and thehydrazone bonds are stabilized by reduction with sodium labelingefficiencies around 60%. The reaction of cytosine with bisulfite in thepresence of an excess of an amine fluorophore leads to transamination atthe N₄ position (Hermanson, 1996). Reaction conditions such as pH, aminefluorophore concentration, and incubation time and temperature affectthe yield of products formed. At high concentrations of the aminefluorophore (3M), transamination can approach 100% yield (Draper andGold, 1980).

Light emissive compounds can be attached to polymers or other materialsby any mechanism known in the art. For instance, functional groups whichare reactive with various light emissive groups include, but are notlimited to, (functional group: reactive group of light emissivecompound) activated ester:amines or anilines; acyl azide:amines oranilines; acyl halide:amines, anilines, alcohols or phenols; acylnitrile: alcohols or phenols; aldehyde:amines or anilines; alkylhalide:amines, anilines, alcohols, phenols or thiols; alkylsulfonate:thiols, alcohols or phenols; anhydride:alcohols, phenols,amines or anilines; aryl halide:thiols; aziridine:thiols or thioethers;carboxylic acid:amines, anilines, alcohols or alkyl halides;diazoalkane:carboxylic acids; epoxide:thiols; haloacetamide:thiols;halotriazine:amines, anilines or phenols; hydrazine:aldehydes orketones; hydroxyamine:aldehydes or ketones; imido ester:amines oranilines; isocyanate:amines or anilines; and isothiocyanate:amines oranilines.

The agent that interacts with the unit of the polymer is selected fromthe group consisting of electromagnetic radiation, a quenching source,and a fluorescence excitation source. “Electromagnetic radiation” asused herein is energy produced by electromagnetic waves. Electromagneticradiation may be in the form of a direct light source or it may beemitted by a light emissive compound such as a donor fluorophore.“Light” as used herein includes electromagnetic energy of any wavelengthincluding visible, infrared and ultraviolet.

As used herein, a quenching source is any entity which alters or iscapable of altering a property of a light emitting source. The propertywhich is altered can include intensity fluorescence lifetime, spectra,fluorescence, or phosphorescence.

A fluorescence excitation source as used herein is any entity capable offluorescing or giving rise to photonic emissions (i.e. electromagneticradiation, directed electric field, temperature, fluorescence,radiation, scintillation, physical contact, or mechanical disruption.)For instance, when the unit is labeled with a radioactive compound theradioactive emission causes molecular excitation of an agent that is ascintillation layer which results in fluorescence.

When a unit of the polymer is exposed to the agent the interactionbetween the two produces a signal. The signal provides information aboutthe polymer. For instance if all units of a particular type, e.g., allof the alanines, of a protein polymer are labeled (intrinsic orextrinsic) with a particular light emissive compound then when a signalcharacteristic of that light emissive compound is detected uponinteraction with the agent the signal signifies that an alanine residueis present at that particular location on the polymer. If each type ofunit e.g., each type of amino acid is labeled with a different lightemissive compound having a distinct light emissive pattern then eachamino acid will interact with the agent to produce a distinct signal. Bydetermining what each signal for each unit of the polymer is, thesequence of units can be determined.

The interaction between the unit and the agent can take a variety offorms, but does not require that the unit and the agent physicallycontact one another. Examples of interactions are as follows. A firsttype of interaction involves the agent being electromagnetic radiationand the unit of the polymer being a light emissive compound (eitherintrinsically or extrinsically labeled with a light emissive compound).When the light emissive unit is contacted with electromagnetic radiation(such as by a laser beam of a suitable wavelength or electromagneticradiation emitted from a donor fluorophore), the electromagneticradiation causes the light emissive compound to emit electromagneticradiation of a specific wavelength. The signal is then measured. Thesignal exhibits a characteristic pattern of light emission and thusindicates that a particular labeled unit of the polymer is present. Inthis case the unit of the polymer is said to “detectably affect theemission of the electromagnetic radiation from the light emissivecompound.”

A second type of interaction involves the agent being a fluorescenceexcitation source and the unit of the polymer being a light emissive ora radioactive compound. When the light emissive unit is contacted withthe fluorescence excitation source, the fluorescence excitation sourcecauses the light emissive compound to emit electromagnetic radiation ofa specific wavelength. When the radioactive unit is contacted with thefluorescence excitation source, the nuclear radiation emitted from theunit causes the fluorescence excitation source to emit electromagneticradiation of a specific wavelength. The signal then is measured.

A variation of these types of interaction involves the presence of athird element of the interaction, a proximate compound which is involvedin generating the signal. For example, a unit may be labeled with alight emissive compound which is a donor fluorophore and a proximatecompound can be an acceptor fluorophore. If the light emissive compoundis placed in an excited state and brought proximate to the acceptorfluorophore, then energy transfer will occur between the donor andacceptor, generating a signal which can be detected as a measure of thepresence of the unit which is light emissive. The light emissivecompound can be placed in the “excited” state by exposing it to light(such as a laser beam) or by exposing it to a fluorescence excitationsource.

Another interaction involves a proximate compound which is a quenchingsource. In this instance, the light emissive unit is caused to emitelectromagnetic radiation by exposing it to light. If the light emissivecompound is placed in proximity to a quenching source, then the signalfrom the light emissive unit will be altered.

A set of interactions parallel to those described above can be createdwherein, however, the light emissive compound is the proximate compoundand the unit is either a quenching source or an acceptor source. Inthese instances the agent is electromagnetic radiation emitted by theproximate compound, and the signal is generated, characteristic of theinteraction between the unit and such radiation, by bringing the unit ininteractive proximity with the proximate compound.

The mechanisms by which each of these interactions produces a detectablesignal is known in the art. For exemplary purposes the mechanism bywhich a donor and acceptor fluorophore interact according to theinvention to produce a detectable signal including practical limitationswhich are known to result from this type of interaction and methods ofreducing or eliminating such limitations is set forth below.

In a preferred embodiment the signal generated by the interactionbetween the unit and the agent results from fluorescence resonanceenergy transfer (FRET) between fluorophores. Either the unit or theproximate compound/agent may be labeled with either the donor oracceptor fluorophore. FRET is the transfer of photonic energy betweenfluorophores. FRET has promise as a tool in characterizing moleculardetail because of its ability to measure distances between two pointsseparated by 10 Å to 100 Å. The angstrom resolution of FRET has beenused in many studies of molecular dynamics and biophysical phenomena(for reviews see Clegg, 1995; Clegg, 1992; Selvin, 1995; and Wu andBrand, 1994). The resolving power of FRET arises because energy transferbetween donor and acceptor fluorophores is dependent on the inversesixth power of the distance between the probes. In practice, thisresolution is about an order of magnitude better than that of thehighest resolution electron microscope.

In order to undergo FRET, the emission spectrum of the donor overlapswith the excitation spectrum of the acceptor. The unit of the polymer isspecifically labeled with an acceptor fluorophore. The agent is a donorfluorophore. A laser is tuned to the excitation wavelength of the donorfluorophore. As the polymer is moved through the channel, the donorfluorophore emits its characteristic wavelength. As the acceptorfluorophore moves into interactive proximity with the donor fluorophore,the acceptor fluorophore is excited by the energy from the donorfluorophore. The consequence of this interaction is that the emission ofthe donor fluorophore is quenched and that of the acceptor fluorophoreis enhanced.

In order to generate an optimal efficient FRET signal for detection, twoconditions should be satisfied. The first condition is efficient donoremission in the absence of acceptors. The second is efficient generationof a change in either donor or acceptor emissions during FRET.

In a model system, both the donor fluorophore and the acceptorfluorophore can give rise to photonic emissions indefinitely. In suchcircumstances, during energy transfer, the decrease in the donoremission is equal to the increase in the acceptor emission. In otherwords, the emission change is identical for both the donor and theacceptor. This is so because for every one donor emission quenchingevent, there is an equal and opposite acceptor emission event. Cantorand Schimmel, 1980 present an intuitive outline of this model system.Consider a system of two fluorophores, a donor and an acceptor. At arange within Förster distance, very weak coupling occurs. Theinteraction between the donor and the acceptor is summarized as:

K_(T) D_(b) + A_(a) ⇌ D_(a) + A_(b) K_(−T)

where D_(a) and A_(a) are the ground singlet states and D_(b) and A_(b)are the first excited singlet states. k_(T) and k_(−T) are the forwardand reverse rates of energy transfer. The above reaction states that fora given donor in the first excited state, it will rapidly lose energy byinternal conversion until it reaches its ground state, D_(a). Since theacceptor absorption energies overlap with the donor emission energies,very weak coupling occurs, allowing resonance energy transfer to takeplace.

As a result of the resonance energy transfer, both the donor (D_(b)) andthe acceptor (A_(b)) are in excited vibrational states. Vibrationalrelaxation rapidly brings these to their respective ground vibrationallevels. Vibrational relaxation is very efficient, with the averagelifetime of an excited vibrational state being only about 10⁻¹⁵ s (Skooget al., 1992). Internal conversion, on the other hand, for fluorescentmolecules is about 10⁻⁹ s. The difference in these rates means that evenwhen k_(T) is very efficient, the reverse reaction (k_(−T)) is unlikelyto occur. In this ideal system, there is thus a shift of the relativepopulation of excited donors and acceptors.

The above analysis sets forth a system utilizing a single acceptor and asingle donor. The same type of interaction may be performed on a systemwhich utilizes many donors and one acceptor. Whenever the acceptor is inits ground singlet state (A_(a)), energy transfer can occur. This meansthat for an acceptor with a lifetime of 1 ns, it can be excited 1×10⁹times per second, a very large rate compared to that of a donorfluorophore under standard illumination (25,000 excitation events/s).Since the maximum number of excitations that an acceptor can undergo ismuch greater than that of one donor fluorophore, multiple donors cantransfer their energy to one acceptor.

Although acceptor excitation to higher energy levels (A_(x)) andsimultaneous donor de-excitation events may be considered to be apotential problem resulting in the interference with signals generatedin a system using multiple donors and a single acceptor the followinganalysis demonstrates that this is not the case. It is improbable thatexcitation to higher energy levels and simultaneous donor de-excitationevents will interfere with signal generation. Acceptor excitation tohigher levels means that there theoretically could be further excitationfrom the acceptor's first singlet state (A_(b)) to the acceptor's highersinglet states (A_(x)). If this were indeed possible, then the

-   -   undergo energy transfer. Such transitions in the acceptor can        only occur if the acceptor absorption energies for a transition        were coincident with the donor emission energies, which is        generally not the case for most molecules. As a consequence,        energy transfer between the donors and the acceptor can only        occur when the acceptor is in its ground singlet state.

In cases of simultaneous donor de-excitation, only one of the donors cantransfer energy to the acceptor. This means that the other donor is notquenched and emits radiatively. If simultaneous events occurredfrequently, then the above scenario for multi-donor quenching would notbe as accurate because a fraction of the donors would be able to emitphotons. The following probability calculations demonstrate that suchevents are statistically infrequent, supporting the use of multi-donorquenching.

The aim of these calculations is to calculate the number of simultaneousdonor de-excitation events for a given amount of time. This number isimportant because the greater the number of overlapping events, thesmaller the percentage of donor energy transfer. For example it ispossible to find the probability of two donor emissions (k=2)simultaneously occurring in one nanosecond for four fluorophores (n=4;thus P_(n,k)=P_(4,2)). Each emission, in this example, lasts for onenanosecond and are represented as blue circles in the grids. Inaddition, each fluorophore emits an average of 25,000 photons/secondrandomly. The variables are defined as n=number of fluorophores,k=number of simultaneous donor emissions, P=probability, C=combination,and t=time. The probability for k simultaneous events occurring in onens for n fluorophores (P_(n,k)) is equal to the probability for ksimultaneous events (P_(k)) multiplied by the number of possiblecombinations that k simultaneous events can occur for n fluorophores(_(n)C_(k)). The total number of simultaneous events is given as the sumof probabilities for all possible values of simultaneous events. Thisgives the probability in the units of (number of events)/ns. The totalnumber of fluorophores undergoing simultaneous emission is thus theprobability for one ns multiplied by the given time (t). It is alsonecessary to consider the probability of greater than two or more donoremissions (P_(4,3) and P_(4,4)) occurring in a given time (t). From thisinformation, the number of donor fluorophores that cannot undergo energytransfer because of simultaneous de-excitation can be derived.

The probability for two simultaneous donor emissions in one nanosecondfor four fluorophores is given as:

$\begin{matrix}{P_{nk} = P_{4,2}} \\{= {\left( P_{k} \right)\left( {{}_{}^{}{}_{}^{}} \right)}} \\{= {P_{k}\frac{n\; t}{{{kt}\left( {n - k} \right)}t}}} \\{= {\left( \frac{25000}{10^{9}} \right)^{2}\left( \frac{4t}{2{t2t}} \right)}} \\{= {3.75 \times 10^{- 9}}}\end{matrix}$

The probability that one ns will contain one emission is 25,000/10⁹. Theprobability that two emissions will occur in the same nanosecond is thesquare (25,000/10⁹)². Since there are four different fluorophores, thereare 6 different possible combinations that give rise to simultaneousemission (4!2!2!). These values multiplied give 3.75.times.10⁻⁹events/ns. This value multiplied by 10⁹ ns yields 3.75 events/s. In thiscase, there are two emissions/event (k=2) and one of these (k−1) cannotundergo energy transfer so the total number of donor emissions that donot undergo FRET is 3.75 events/s.

The above simple calculation can be applied to a system with a greaternumber of fluorophores. In this case, one thousand fluorophores areconsidered (n=1000). In a similar fashion, P_(1000,2) is calculated tobe 3.122×10⁻⁴ events/ns. The number of donor emissions that cannotundergo energy transfer is 3.122×10⁵. In some cases, three simultaneousdonor emissions can occur. The probability for these events(P_(10000,3)) is calculated:

$= {{\left( \frac{25000}{10^{9}} \right)^{3}\left( \frac{1000t}{3t\; 997\; t} \right)} = {2.596 \times 10^{- 6}}}$

The number of donor emissions that cannot occur is(3-1)(10⁹)(2.596×10⁻⁶), or 5.192×10³. In a similar fashion, calculationsfor four simultaneous emissions or greater need to be included in thenet number of donor fluorophores that cannot undergo energy transfer.This number is expressed as the following equation:

k − n $\begin{matrix}{= {{\left( {2 - 1} \right)P_{n{.2}}t} = {{\left( {3 - 1} \right)P_{n{.3}}t} + \ldots + {\left( {n - 1} \right)P_{n.n}t}}}} \\{= {\sum{\left( {k - 1} \right)P_{n,k}t}}}\end{matrix}$ k − 2

Solving the above summations yields 3.174×10⁵ donor emissions that donot undergo energy transfer. The donor emission is calculated from thefollowing values: 25000 photons/s per fluorophore, 1000 fluorophores,and a 1 second time interval. This means that 2.5×10⁷ photons areemitted per second. The conclusion is that 98.7% of donor events canundergo energy transfer.

The conclusion from the above discussion of a multiple donor/singleacceptor system is that one acceptor can undergo energy transfer withmultiple donors. The limitations that were considered were the abilityto excite an acceptor to higher energy levels and also the limitationsof simultaneous donor de-excitation. In donor emissions that occurconcurrently, only one of the de-excitations would be able to undergoenergy transfer with the acceptor. If this occurred often, then theacceptor would not be able to undergo energy transfer with all donoremission events. Probability calculations show that for a one thousanddonor/one acceptor system under standard epiillumination conditions,98.7% of donor emission events can undergo energy transfer with anacceptor. The overall conclusion from these detailed analyses is that amulti-donor system/one acceptor system can indeed exist and that asingle acceptor can undergo energy transfer with a large number of donormolecules.

The following description sets forth how conditions can be optimized forthe donor and the acceptor fluorophores of the multi-donor/one acceptorsystem. In order to optimize the system two potential problems which mayarise when this system is actually performed should be considered. Theseare photobleaching and solvent quenching (Rost, 1990; Menter et al.,1978; Giloh and Sedat, 1982; Vaughan and Weber, 1970; Guilbault, 1973;Udenfriend 1962; Pringsheim, 1963). These effects may limit the numberof excitation cycles a fluorophore can undergo. Whereas in an idealsystem a fluorophore can undergo infinite excitation cycles, inpractice, the actual number of cycles is limited to a finite numberdepending on both the solvent conditions and intensity of the excitationlight. The signal produced in a multiple donor/one acceptor system,however, can be optimized under certain experimental conditions.

The theory for optimizing and generating an efficient signal follows.The rationale is that as long as model conditions are simulated as closeas possible, signals are generated. Recall that ideal fluorophores canundergo infinite cycles of excitation. If realistic fluorophores behavein a similar fashion, then any number of fluorophores can be detected aslong as temporal integration of photonic emissions is sufficient. Onemethod for achieving close to ideal conditions is by immobilizingfluorophores in a solid medium. Embedding fluorophores in solid mediacan abolish both photobleaching and quenching (Haughland, 1996; Garlandand Moore, 1979; Rost, 1991) and that certain types of fluorophores areespecially stable (such as ferulic acid in plant cell walls, uranylglass, and Acriflavine) (Rost, 1991). This will dramatically increasethe number of possible excitation cycles. Despite being embedded in asolid medium, donor fluorophores can generate fluorescent signals forseveral hours (Rost, 1991) and can still undergo FRET with acceptors(Stryer, 1978; Cantor and Schimmel, 1980) and vise versa. Furthermore itis known that the acceptor can undergo resonance energy transfer withdonors for several hours in solvent conditions (Wittwer, 1997; Lee etal., 1994; Uchiyama et al., 1996; Livak et al., 1997). The ability foran acceptor to quench a donor that has a stable signal translates intothe ability to generate highly efficient signals.

It has been shown that an acceptor can undergo energy transfer with adonor for an extended period of time in solvent conditions (Wittwer,1997; Lee et al., 1994; Uchiyama et al., 1996; Livak et al., 1997). Theevidence arises from experiments done on peptide and nucleic acidcleavage assays. In these assays, a particular substrate is labeled ateither end with a donor and an acceptor fluorophore. Since the length ofthe substrate is within range of the Förster distance of thedonor-acceptor pair, the fluorescence of the donor is quenched.Monitoring the donor fluorescence allows a light based assay ofcleavage. In this manner, an increase in donor fluorescence is directlyproportional to the cleavage activity of the particular enzyme. Assaysof this type, commonly called fluorometric cleavage assays, have beenused to study a number of systems including HIV proteases (Matayoshi etal., 1990; Toth and Marshall, 1990), neutral proteases (Ng et al.,1989), EcoRV restriction endonucleases (Erskine and Halford, 1994),PaeR7 endonuclease (Ghosh et al., 1994), DNA polymerase 15′-3′exonuclease activity (Wittwer et al., 1997; Livak, 1997), thermolysin(Yang and Van Wart, 1994), and papain (Garcia-Echeverria and Rich,1992). The relevance of these experiments is important because theycontain evidence that an acceptor can quench a donor for extendedperiods of time. The controls in fluorometric cleavage assays includethe monitoring of uncleaved substrates (in the absence of the cleavageenzymes) for the duration of the experiment. For especially lengthyexperiments, these controls need to be monitored for an extended periodof time. The lack of increase in donor fluorescence in these controlsdemonstrates directly that an acceptor can quench a donor for asignificant length of time.

The acceptor maintains its ability to transfer energy for an extendedperiod of time because its energy states remain unchanged. Energytransfer occurs when the acceptor absorption energies are coincidentwith the donor emission energies. The result of energy transfer is aground singlet state (A_(a)) to excited singlet state (A_(b))conversion. The acceptor loses energy through internal conversion,either fluorescent or non-radiative. The studies cited above demonstratethat despite non-ideal experimental conditions, the acceptors maintaintheir relative energy levels and hence their ability to undergo energytransfer.

The number of excitation cycles that a fluorophore generally undergoesin a solution is roughly 35,000 cycles (Rost, 1991). The number offluorophore excitation cycles in solution, however, is limited by thesolvent effects discussed above such as oxygen quenching, collisionalquenching, and excited state reactions. Solvent conditions can beadjusted so that the number of excitation cycles is optimized(Haughland, 1996). Various chemical methods are used to optimizeconditions for detection of acceptor fluorescence. Sodium azide (NaN₃),sodium iodide (NaI), dithiothreitol (DTT), dithioerythritol (DTE),sodium dithionate, n-propyl gallate, ascorbic acid, and polyvinylalcohol (PVA) all have been found effective with various fluorophores(Bock et al., 1985; Johnson et al., 1982; Picciolo and Kaplan, 1984;Gill, 1979; Giloh and Sedat, 1982; Valnes and Brandtzaeg, 1985).b-mercaptoethanol, sodium nitroprusside, and incorporation of electrondonors and molecules with SH groups have also been found to be effective(Franklin and Filion, 1985; Spatz and Grabig, 1983; Hamada and Fujita,1983). In addition, commercial reagents for reducing solutionfluorophore fading are available. SlowFade (Molecular Probes, Oreg.)formulation reduces the fading rate of fluorescein to almost zero.Because it provides a nearly constant emission intensity fromfluorescein, the SlowFade reagent is especially useful for quantitativemeasurements and applications that employ a confocal laser scanningmicroscope, in which the excitation intensities can be extreme andprolonged. For a system under the proposed optimized conditions, bothinitial donor emission and the change in donor emission in the presenceof acceptors have efficiencies close to that of ideal conditions.

A “detectable signal” as used herein is any type of signal which can besensed by conventional technology. The signal produced depends on thetype of agent or station as well as the unit and the proximate compoundIf present. In one embodiment the signal is electromagnetic radiationresulting from light emission by a labeled (intrinsic or extrinsic) unitof the polymer or by the proximate compound. In another embodiment thesignal is fluorescence resulting from an interaction of a radioactiveemission with a scintillation layer. The detected signals may be storedin a database for analysis. One method for analyzing the stored signalsis by comparing the stored signals to a pattern of signals from anotherpolymer to determine the relatedness of the two polymers. Another methodfor analysis of the detected signals is by comparing the detectedsignals to a known pattern of signals characteristic of a known polymerto determine the relatedness of the polymer being analyzed to the knownpolymer. Comparison of signals is discussed in more detail below.

More than one detectable signal may be detected. For instance a firstindividual unit may interact with the agent or station to produce afirst detectable signal and a second individual unit may interact withthe agent or station to produce a second detectable signal differentfrom the first detectable signal. This enables more than one type ofunit to be detected on a single polymer.

The detectable signal is produced at a station. A “station” as usedherein is a region where a portion of the polymer to be detected, e.g.the unit, is exposed to, in order to produce a signal or polymerdependent impulse. The station may be composed of any material includinga gas. Preferably the station is a non-liquid material. “Non-liquid” hasits ordinary meaning in the art. A liquid is a non-solid, non-gaseousmaterial characterized by free movement of its constituent moleculesamong themselves but without the tendency to separate. In anotherpreferred embodiment the station is a solid material. In one embodimentwhen the unit interacts with an agent the station is an interactionstation. The station may also be a signal generation station, which isdiscussed in more detail below. As used herein an “interaction stationor site” is a region where a unit of the polymer and the agent can bepositioned with respect to one another in close enough proximity wherebythey can interact. The interaction station for fluorophores, forexample, is that region where they are close enough so that theyenergetically interact to produce a signal.

The interaction station in a preferred embodiment is a region of ananochannel where a localized agent, such as an acceptor fluorophore,attached to the wall forming the channel, can interact with a polymerpassing through the channel. The point where the polymer passes thelocalized region of agent is the interaction station. As each labeledunit of the polymer passes by the agent a detectable signal isgenerated. The agent may be localized within the region of the channelin a variety of ways. For instance the agent may be embedded in thematerial that forms the wall of the channel or the agent may be attachedto the surface of the wall material. Alternatively the agent may be alight source which is positioned a distance from the channel but whichis capable of transporting light directly to a region of the channelthrough a waveguide. An apparatus may also be used in which multiplepolymers are transported through multiple channels. These and otherrelated embodiments of the invention are discussed in more detail below.The movement of the polymer may be assisted by the use of a groove orring to guide the polymer.

Other arrangements for creating interaction stations are embraced by theinvention. For example, a polymer can be passed through a molecularmotor tethered to the surface of a wall or embedded in a wall, therebybringing units of the polymer sequentially to a specific location,preferably in interactive proximity to a proximate agent, therebydefining an interaction station. A molecular motor is a compound such aspolymerase, helicase, or actin which interacts with the polymer and istransported along the length of the polymer past each unit. Likewise,the polymer can be held from movement and a reader can be moved alongthe polymer, the reader having attached to it the agent. For instancethe agent may be held within a scanning tip that is guided along thelength of the polymer. Interaction stations then are created as theagent is moved into interactive proximity to each unit of the polymer.

Once the signal is generated it can then be detected. The particulartype of detection means will depend on the type of signal generatedwhich of course will depend on the type of interaction which occursbetween the unit and the agent. Many interactions involved in the methodof the invention will produce an electromagnetic radiation signal. Manymethods are known in the art for detecting electromagnetic radiationsignals, including two- and three-dimensional imaging systems.

Three-dimensional imaging systems for imaging fluorescence in biologicalsystems has recently been described. Dickson et. al., have describedthree-dimensional imaging of single molecules in the pores of a gel.Dickson et. al., Science, 274:966-969 (1996). Dickson et al. examine theeffects of confined environments on Brownian motion of singlefluorescent molecules. Single molecules of free fluorescent dye andfluorescent dye bound to protein were trapped in polyacrylamide (PAA)gels. Nile red was incorporated into polyacrylamide gels having pores onthe order of 2 nm. The fluorophore gels were excited with an evanescentwave generated by total internal reflection (TIR) to detect movement orimmobilization of single molecules within the pores of the gels. The TIRmethod produces three-dimensional information about the molecules in thepores by using the exponential fall-off in excitation intensity todetermine the distance of the fluorophore from the interface. Thediscrepancy between observed motion and theoretical Brownian motion wasstudied by both cases. The Brownian motion for free fluorophores wasreduced by a factor of 10⁴. The labeled proteins remained completelystationary in space. By direct analogy, the spatial confinement of thenanochannels should limit or eliminate the Brownian motion of thelabeled DNA in nanochannel FRET sequencing. This would allow a stableand predictable passage of the DNA through the nanochannels.

An example of a microscopy system useful according to the methods of theinvention is provided in Ishijima et al., Cell, 92:161-171 (1998).

Two-dimensional imaging systems are important because they have, amongother parameters, low noise, high quantum efficiency, properpixel-to-image correlation, and efficient processing times. An exampleof a device useful for detecting signals is a two-dimensionalfluorescence imaging system which detects electromagnetic radiation inthe fluorescent wavelength range.

There are three categories of fluorescence imaging devices based on thetype of fluorescence signal measured, either intensity, lifetime, orspectra. Intensity signals can be captured by a variety of methodsincluding charge coupled device (CCD) camera, streak cameras, andsilicon diode arrays. Of these methods, the most common is the CCDcamera because of its wide commercial applications. The streak cameraoffers a superior temporal resolution down to the femtosecond. Silicondiode arrays have superior dynamic range, signal to noise ratios, andtemporal resolution (common frame rates at 1000/s), but have largerpicture elements (25 μM×500 μM as opposed to 20 μM×20 μM for a CCD).Each of the devices may be used with the methods of the invention butCCD cameras are preferred.

Lifetime and spectral imaging are performed using a combination ofinstruments including gated image intensifiers, pulsed lasers, and CCDcameras. Time-gated methods, which are lifetime-related techniques,involve temporally discriminating fluorescence signals from backgroundand autofluorescence. Periasamy et. al., which is hereby incorporated byreference provides a review of Time-gated fluorescence microscopy forclinical imaging. Periasamy et. al., Microscopy and Analysis, 33-35(1995). Lifetime imaging uses time-gating or phase-modulation techniquesto determine fluorescence lifetime. Spectral imaging determines theemission spectrum at each pixel. Time-gated and lifetime imagingtechniques offer many advantages to simple CCD imaging such as bettersignal-to-noise ratios and greater dye specificity and thus also arepreferred devices for the detection method of the invention.

The first type of imaging technique, intensity imaging generallyinvolves the use of a CCD or ICCD camera to independently captureintensity signals. This is a desirable method for detecting signalsaccording to the methods of the invention because of its simplicity. CCDand ICCD cameras can be readily purchased from a number of suppliers(i.e. Photometrics, Hamamatsu, Princeton Instruments).

CCDs are two-dimensional silicon matrices that have many light sensitiveelements called pixels that can hold electronic charges generated byphoton interaction. Exposure of the CCD to photonic fluorescent emissioncauses accretion of charge in the individual pixels. After an exposureis complete, entire rows of accumulated charge are transported towards aserial CCD register. In the serial register, individual pixel chargepackets are transported to a read-out amplifier that creates a signalproportional to the amount of charge. Each row of the CCD is read in asimilar fashion until the image is successfully converted to atwo-dimensional series of signals. Charge transfer between the CCD andthe serial CCD register is extremely efficient (99.9999% efficient). Theoutput amplifier creates a linear response to the measured analogsignals and the image is digitized between 8 and 16 bits.

Upon read-out of the image, there are two options that CCD camerasusually employ to modify images. These are subarray read-out andbinning, or charge-grouping. A programmed CCD camera can selectivelyprocess pixels in a defined region of interest. The advantage ofdefining regions of interest is that it allows faster image read-out tooccur. The time to process and digitize each pixel is fixed, so thatsmaller desired regions allow for higher frame rates. Smaller regionsare possible from subarray read-out. For instance a 100×100 pixel in a1024×1024 pixel CCD may be read at a frame rate of 100 frames/s at apixel read frequency of 1 MHZ. Binning, which is a combination ofsmaller pixels into one effective larger pixel for faster readout times,on the other hand, allows combination of charge from several pixels.During binning, the CCD operates at reduced resolution for increaseddynamic range and a higher frame rate. The dynamic range is the ratio ofthe largest signal a CCD can detect linearly to the basal readout noise.For example, a 1024×1024 pixel CCD binned 4×4 yields a 256×256 imagewhich has large pixels that are effectively 16 times larger than theunbinned version. The image is also read-out at 16 times the rate. Thespecific operational mode of the CCD is dictated by the components ofthe camera and a host computer.

To understand the control of the CCD, there are three components of atypical CCD that need to be discussed, including the camera controller,electronics unit, and camera head. The controller acts as theintermediary processor of input and output between the host computer andthe camera. The controller contains logic which causes the camera to acton certain host commands. In addition it also relays digitized pixeldata to the computer. Internally, the controller has sequences necessaryfor coordination of the CCD phases and timing of the analog processingunits. The electronics unit convert digital commands into activeclocking signals and sequences. Furthermore, the unit contains the A/Dconverter. Digitizers from 8 to 16 bits are commonly used. The camerahead contains the CCD and often a cooling device. Liquid nitrogen orPeltier cooling are common.

With a basic understanding of the operation of a CCD camera, performanceis the most important consideration for the camera. Specifically, theseparameters are noise, linearity, quantum efficiency, and temporalresolution. There are four components of noise that are important. Theyare dark current, read noise, shot noise, and lamp noise. Dark currentis the leakage current within the CCD. The charge accumulates even inthe dark, hence the term. The lower the CCD temperature, the lower thedark current. Thermoelectric coolers or liquid nitrogen can lower theCCD temperature to reduce the dark current. Temperatures of −120° C. canreduce the dark current by several orders of magnitude. Multi-pinphasing (MPP) is a new CCD technology that can reduce dark current by afactor of 100 or more. Read noise is the stochastic electronic chargegenerated at higher frame rates arising from the output preamplifier. Asthe collection rate increases, so does the read noise. Read noise can besignificantly reduced when the temperature is lowered below −60°. Shotnoise, also commonly called photonic noise, arises because of thequantum nature of light. It is the square root of the photonic signal.At low light levels, the relative shot noise is high and often masks thedesired signal. Lamp noise is due to intensity fluctuations in theilluminating source and can be controlled by using stable powersupplies. Lamp noise is very often overlooked in quantitativeapplications of CCD cameras.

Photometric linearity becomes especially important during quantitativefluorescence microscopy. For use in the methods of the invention,concern for linearity is not overly important because in general linearoperators are not being applied to the CCD data. Since the chargegeneration mechanism of a CCD is intrinsically linear, the output signalis precisely proportional to the charge. The electronics between the CCDand the digitizer provide room for deviations. High scientific gradeCCDs usually exhibit linearity with less than 0.1% deviation. In mostcases, the linearity is difficult to measure because it is more linearthan techniques used to test it.

Quantum efficiency is the fraction of photons that reach the CCD thatinteract to generate electron-hole pairs in the CCD. Quantumefficiencies range from 10% on low-grade CCDs to as high as 80% on backilluminated scientific grade CCDs. There are also spectral variances ofthe quantum efficiency, with peaks occurring usually in the visiblewavelength. Back illuminated CCDs have much greater quantum efficienciesat all spectral positions.

Temporal resolution of a CCD camera becomes important when discussingthe possible frame rate of the camera. Exposure times vary for givenapplications and the exposure time can be adjusted by the number offluorophores used and the intensity of the excitation light. Thelimitation to the temporal resolution in a given CCD camera is afunction of the analog-to-digital converter operating frequency and alsothe image size. For example, an A/D converter operating at 1 MHZ canread 100 frames of a 100×100 image in one second. In addition, thehigher the dynamic range desired, the slower the read frequency. Forexample, it is difficult for current CCD cameras digitizing at 14 bitsper pixel to operate above 1 MHZ. Use of multiple serial registerdevices overcomes the temporal limitations imposed by one A/D converter.The speed increase with such a device is proportional to the number ofavailable output channels. A 14 bit 100×100 pixel image acquired by aCCD camera with 4 output registers operating at 1 MHZ can acquire 400frames per second. The maximum rate limitation that cannot be overcomeis dictated by the pixel read-out times, which vary from 20 μs to 500ns. For a 100×100 pixel image, even the slowest pixel read-out time canallow 500 frames/s. Hence, read-out times are rarely a limitation on theframe rate, but rather it is the A/D converter that is limiting. Asevidence of this, frame rates of 2000/s have been possible with a128×128 array (Ichikawa et al., 1992).

A CCD camera which is particularly useful in the methods of theinvention is a large pixel, low noise, and short scan time camera. Largepixel sizes have greater well capacities and allow for greatercollection of photons and hence a greater maximal signal-to-noise ratio(SNR). The basis of intensity change discrimination relies on a largeSNR, as is to be discussed shortly. Accordingly, low noise aids inimproving the SNR. MPP type CCDs with smaller well capacity and lowerdynamic range are not well suited. The temporal resolution is high fromthe short scan time, allowing a high volume data stream that should bestorable in real time in the computer RAM or on the hard drive. Thequantum efficiency should be reasonable. The linearity of the CCD is notcritical, but assumed to be scientific grade (0.1%). The dynamic rangeis not critical because the donor fluorophores that are images areapproximately the same intensity.

An intensified CCDs (ICCDs) camera consists of a photocathode,microchannel plate (MCP), and a phosphor screen in addition to the CCDcamera. Fluorescence light impinges on the photocathode, releasingphotoelectrons into the MCP. The MCP is a secondary electron multiplierconsisting of an array of millions of glass capillaries (channels) fusedinto the form of a thin disc. When photoelectrons are incident upon thechannels, secondary electrons are produced. Passage of the electronsthrough the channels produces an amplification that is directlycontrolled as the gain of the instrument. Each channel of the MCPcorresponds to a picture element. The output of the MCP is focused ontoa phosphor screen where electrons exiting from the MCP strike. Theoptical image is reproduced on the phosphor screen and is captured by aCCD camera.

In some instances, it is preferred to use a ICCD. The addition of theimage intensifier offers high speed gating and high gain. Gating isprobably the most important aspect of the ICCD. It is the electronicshutter action produced by controlling the input voltage to the imageintensifier. At present, time-resolved imaging methods are possible withnanosecond and sub-nanosecond gating times (Thomas et al., 1992). Gatingallows for greater signal detection because of temporal discriminationof background signals. Examples of temporal discrimination andenhancement of signal-to-noise ratios is seen in time-gated fluorescencemicroscopy and the pulse method of fluorescence lifetime imagingmicroscopy. The gain of the image intensifier is used to increase thesensitivity of the camera and also serves as a tool in thephase-modulated method of fluorescence lifetime imaging spectroscopy.The electronic gain can be as high as 10, 000:1. This allows faintoptical signals to be amplified over read noise.

Single photon events can be detected with an ICCD. Single fluorophoreimaging, for instance, has been achieved by Sase et al., 1995 using aCCD camera, image intensifier, and an epifluorescence microscope. In“Real Time Imaging of Single Fluorophores on Moving Actin with anEpifluorescence Microscope,” Sase et al. demonstrate that singlefluorophores can be imaged in real time with a high detectionefficiency. Other methods that have achieved solution single moleculesensitivity include fluorescence correlation spectroscopy (Eigen andRigler, 1994; Kinjo and Rigler, 1995), far-field confocal microscopy(Nie et al., 1994), cryogenic fluorescence spectroscopy (Kartha et al.,1995), single molecule photon burst counting (Haab and Mathies, 1995;Castro and Shera, 1995; Goodwin et al., 1995; Peck et al., 1989; Nguyenand Keller 1987; Lee et al., 1994; Chen and Dovichi, 1996; Shera et al.,1990; Soper et al., 1992), two-photon excited fluorescence (Mertz,1995), and electrochemical detection (Fan and Bard, 1995).

A numerical SNR value can be calculated based on the desired aspects ofa CCD camera. For example, a CCD that satisfies the criteria set outabove is an EEV 05-20 CCD (Princeton Instruments, (Princeton, N.J.)which has the specifications set forth in Table 1:

TABLE 1 CCDformat 1152 × 770  dynamic range, bits 14 to 17 pixel size,μm 22.5 × 22.5 non-linearity, % <1 (16 bits) full well 500 dark chargeat −120° C., <1 capacity, ke- electrons/pixel-hour readout noise, e-4-6, 50 kHz quantum eff., % peak 40 22,500 kHz

In order to calculate a SNR, an equation that considers the variousnoise values is needed. In any detection system, there is always a basallevel of noise that may hinder detection of signals that are weak andintermittent. Intuitively, the larger the desired signal, the lessimportant the basal level of noise becomes. A larger signal and hence alarger SNR can be attained by increasing either the number offluorophores or the excitation intensity. The equation for calculatingthe SNR is

${SNR} = \frac{PE}{\left( \sqrt{N_{shot}^{2} + N_{dark}^{2} + N_{read}^{2} + N_{lamp}^{2}} \right)}$${PE} = \frac{{IE}\; \rho \; {tENG}}{hv}$

PE represents the number of photoelectrons emitted from the detector andis related to the efficiency (E) of the detector system. The higher theefficiency (E), the greater the number of photoelectrons emitted for agiven number of photons. PE is also related to the number offluorophores (N), pre-amplifier gain (G), integration time (t),intensity of light (I), molar extinction coefficient of the fluorophoreE), and a fluorescence constant specific for the chosen fluorophore (p).N_(shot) represents the noise due to random fluctuations of thefluorophore emission and is related to the magnitude of the signalproduced. The larger the signal, the larger the shot noise. N_(dark) andN_(read) are dark noise and read noise. N_(lamp) is the noise generatedfrom the illuminating source. It is important to note that N_(lamp) canhave large influences in the SNR. An illumination source that has a 1%intensity fluctuation can have a SNR only as large as 100:1. In thiscase, the lamp noise is minimized by using a stable power supply. Forexample, commercially available (Uniphase) helium-neon lasers withmodified power supplies have intensity fluctuations of less than 0.002%of the total intensity. Furthermore, a tungsten filament lamp can beequipped with a stable power supply so that the output of the bulbfluctuates less than 0.001%. Table 2 lists the values for the variablesand provides the reason the particular values were chosen.

TABLE 2 variable value reason I 30 W/cm² intensity of 2 W laser is givenby P/A. A is the beam area. Intensity of laser is 64 W/cm2 ε 91,000 1/Mcm molar extinction coefficient for fluorescein ρ 3.8 × 10⁻²¹ M cm³constant for fluorescence emission of fluorescein t 0.010 s integrationtime for CCD operating at 100 frames/s E 0.025 collection efficiency ofsystem including filters, objective, and quantum efficiency of CCD N1000 reasonable number of donor fluorophores/pixel G 60 typicalpre-amplifier gain; gain for ICCD can be as high as 10,000:1 h 6.6261 ×10⁻³⁴ J s Planck's constant v 6.1224 × 10¹⁴ × s⁻¹ C = vλ; λ = 490 nm(excitation of fluorescein) N_(shot) 632e⁻¹ N_(shot) = PE N_(lamp)  8e⁻¹0.002% intensity fluctuation N_(read)  40e⁻¹ estimation for EEV 05-20CCD at high readout speeds N_(dark) ~0 value for EEV 05-20 CCD at −120°C. SNR 631:1 from the above equation

A SNR of 631:1 is high. A high SNR ensures efficient detection of bothintensity and intensity changes, or in other words, donor emission inthe absence and presence of an acceptor. For example, a SNR of 631:1means that there is a 66% confidence of detecting a 0.158% (1/632×100)change in the signal. There is a 95% confidence in detecting a 0.316%change and a 99.9% confidence for a 0.475% change. The larger theintensity change, the greater chance of detection. A higher baseline SNRin the absence of any intensity changes allows for greater confidenceintervals for a given percentage intensity change. In order to maximizethe confidence of detection of a signal change, it is important togenerate large percentage changes in the presence of acceptors.

One method for generating a large percentage change is to clustermultiple donor fluorophores around the interaction station, e.g., in aconcentric ring which the polymer can pass through, ensuring that alldonor fluorophores will undergo energy transfer with the acceptor. Anappropriate width of such a concentric ring of donor fluorophores can bedetermined by the rate of emission of the donors and also the Försterdistance of the donor-acceptor pair. Typical changes in intensity uponacceptor passage range from 30% to 50%, which correspond to anunequivocal 100% confidence.

Before the CCD camera can process a signal each signal generated must becaptured by pixels of the detector system. Each pixel should be capableof capturing a signal from a single interaction station and should havethe ability to detect transient changes in the signal. The area of aninteraction station, e.g., the localized region of agent on ananochannel, detected by one pixel is determined by the pixel size onthe detector, the magnification of the image, and also the diffractionlimit of the wavelengths to be measured. The relationship between themeasured area, detector pixel size, and magnification is given by thefollowing equation.

a=2d/M

The size of the area to be measured is given by a² where a is the edgelength. The edge length of the detector pixel is given by d. Themagnification is given by M. Using convention values of these variables,with d=15 μM and M−60×, the edge length of a measured area turns out tobe 500 nm, well within range of the resolving power of a 500 nmwavelength signal. The resolving power for a microscope sample underepiillumination is presented in the following equation (Matsumoto,1993).

R _(f)=0.61λ/NA

R_(f) is the minimum distance between two bright points that can beresolved. λ is the wavelength of the donor fluorescent emission. NA isthe numerical aperture of the microscope objective. The highestnumerical aperture is desired because of the inverse relationshipbetween NA and the resolving power. NA is given by the following classiccriterion for lens selection (Taylor and Salmon, 1989):

NA=n sin Θ

n is the index of refraction of the immersion medium. It is oftendesirable to use a higher index medium such as oil. Θ is the anglebetween the optical axis and the greatest marginal ray entering thelens. For quality microscope objectives at high powers, the numericalaperture can be as high as 1.4. From the above two equations, theresolving power for a 500 nm emission signal and a 1.4 NA lens becomes218 nm.

The conclusion from the above calculations is that two adjacent pixelson the detection unit can each detect signals from their respectiveinteraction stations without confusion of the origins of the signals. Bycarefully calculating the magnification and the pixel sizecross-interference between pixels can be avoided. For example if thesmallest pixel size (d=6 μM) and the largest magnification (M=100×),were used a 120 nm edge length on the detected area is achieved. Sincethe resolving power of the system remains constant at 218 nm,interference of signals would exist. This can be avoided by performingcalculations prior to defining the experimental set up. Hence, byadjusting the magnification and the pixel size on the detection system,an optimal number can be reached where the measured area matches thediffraction limit. This can be determined from a combination of theabove two formulas by setting the edge length equal to or greater thanthe resolving power of the detection system:

2d/M>0.61%/NA

the density of the interaction stations can be varied so that only oneinteraction station corresponds to one pixel of the detector system. Forexample, if a 4×10⁶ nanochannels/cm² plate is used, 0.01 nanochannels(interaction stations) are found per pixel using a 60× magnification anda 15 μM pixel size. The interaction station density can be adjusted evenlower, e.g., an average minimum inter-pore distance of 48 μM is alsowithin the appropriate range.

In addition to intensity imaging both time-gated fluorescence microscopyand fluorescence lifetime imaging may be used to detect signalsaccording to the methods of the invention. Time-gated fluorescencemicroscopy and fluorescence lifetime imaging are more involved methodsthat have advantages such as temporal discrimination of fluorescentsignals and better signal-to-noise ratios than intensity imaging.

Time-gating is desirable if there is significant background scatteringor autofluorescence. Background light scattering is a problem when thescattered wavelengths are equal to the emission wavelengths of thedesired fluorophores. Scattering effects can be avoided by using afluorophore with a large Stokes shift so that the scattered wavelengthsare shorter than the detected wavelength. In this case, the scatteredlight can be eliminated by using optical filters. On the other hand,autofluorescence is a common problem affecting essentially all studiesemploying fluorescence microscopy. Autofluorescence can arise fromsolvents, solutes, and the optical components of the microscope system.Autofluorescence decreases the signal-to-noise ratio of detection. Thisis the case even though there have been many improvements in the variouscomponents of the detector systems (Periasamy and Herman, 1994).

Time-gated fluorescence microscopy (TGFM) utilizes differingfluorescence lifetimes to make a distinction between autofluorescenceand fluorescence. Lifetimes of fluorescent dyes can be chosen to belonger than that of autofluorescence. Short lived autofluorescencedecays to zero in less than 1 μs whereas long lived fluorescence candemonstrate lifetimes from 1 μs to 10 ms (ex. europium chelates,lanthanide chelates). Excitation of the sample is done with a briefintensity pulse, shorter than the lifetime of either theautofluorescence or fluorescence. Exponential decay follows. Ifmeasurements are made only after the decay of the fluorescent signal,then the longer lived signals are measured with a greater sensitivity.

An example of an imaging apparatus for TGFM is provided in Periasamy,1995. A fluorescent microscope (Nikon) with epiillumination capabilitiesand a continuous wave (CW) laser (Coherent or Spectra-Physics) are shownin the reference, emitting at the desired excitation wavelength. Theexcitation laser light is chopped by a chopper with a chopper control tocreate laser pulses with defined pulse widths. The intensity of thelight is controlled using a variable neutral density filter (OmegaOptics). To delay the time of measurement, a delay pulse generator isused to generate a signal for controlling a high frequency gated imageintensifier (Hamamatsu) or a chopper in the emission light path. A CCDcamera (Princeton Instruments or Photometrics) is used to collect thesignals.

The fluorescence lifetime represents the average amount of time amolecule remains in the excited state prior to its return to groundstate. There are two methods for the measurement of fluorescencelifetimes. These are the pulse method and the phase-modulation method(Lakowicz, 1986; McGown, 1989; Gratton and Limkema, 1983). In the pulsemethod, the sample is excited with a brief pulse of light and thetime-dependent fluorescent decay is measured. In the phase-modulationmethod, the sample is excited with a sinusoidally modulated light. Thephase shift and demodulation is used to calculate the lifetime. Untilrecently, lifetime measurements were only used with cuvette samples. Inthe past five years, there has been development of methods that combinemicroscopic two-dimensional resolution and high-resolution lifetimemeasurements (Rodgers and Firey, 1985; Wang et al., 1990; Morgan et al.,1990; Clegg et al., 1991; Lakowicz and Bemdt, 1991; Buurman et al.,1992; van de Ven and Gratton, 1992; Oida et al., 1993). The developmentof fluorescence lifetime imaging microscopy (FLIM) has allowed thedetailed study of location and environment of fluorescent labels incells and other microscopic samples. In the following, the advantages,theory, and applications of FLIM are discussed.

Fluorescence lifetime measurements have been used for a variety ofreasons including specificity, sensitivity, quantitation, and hightemporal resolution (Wang et al., 1996). Measurement of lifetimesprovide high specificity because fluorescent molecules have distinctlifetimes. In comparison to absorption and emissions, lifetimes providegreater discrimination of molecules. Lifetimes can also be carried outon small amounts of molecules, leading to similar sensitivities asintensity measurements. Quantitation of molecules through lifetimesprovide a true measurement because fluorescence lifetime is directlyrelated to the fluorescence quantum yield of the fluorophore. Lastly,lifetimes can be used to detect temporal events that occur on the timescale of biomolecular processes, usually between a picosecond and amicrosecond.

There are two methods of determining lifetimes. The pulse method isdescribed first (Lakowicz, 1986). Consider an short pulse of lightexciting a population of fluorophores. The fluorescent signal of theexcited molecules decays with time in a first order manner, given as thefollowing exponential function:

I(t)=Ae ^(−t/τ)

a is an arbitrary constant, t is the time, and τ is the fluorescentlifetime. Intuitively, the fluorescent lifetime is the time required forthe intensity to decay to 1/e of the original value, a decay of 63%. Oneof the methods to experimentally measure the lifetime is to use apulse-sampling method (Herman et al., 1996). D₁ and D₂ are collected onconsecutive frames. Frames are analyzed with a lifetime equation and aFIGS. 2 and 2-D lifetime array is generated. Following each excitationevent, the multichannel plate gated image intensifier (MCP-GII) attachedto the CCD camera is turned on for a very brief interval (i.e. 4 ns) atsome time interval (t₁) after the exciting pulse. The emission isacquired on a CCD that is continually on. The identical process isrepeated for a large number of times to capture a sufficient signal onone frame of the CCD. After a sufficient signal at t₁ is generated, theCCD is read out and the gate window with respect to the excitation pulse(t₂) is shifted and the whole process is repeated. Interpretation of thetwo frames with a pixel by pixel analysis gives the lifetime of theimage at each point.

Wang et al., 1996 describe the apparatus for pulse FLIM as consisting offive main components: 1) pulsed light source; 2) image detection system(gated image intensifier and CCD camera; 3) timing control unit; 4) andfluorescence microscope. The system is identical to the apparatusdescribed for time-gated fluorescence microscopy (TGFM) described above,except for the pulsed light source which is a picosecond pulsed lightsource (Coherent) consisting of a mode-locked YAG laser, a dye laserwith a third harmonic generator, and a cavity dumper. Picosecond pulseshaving tunable wavelengths from UV to IR. Rates from single shot to 76MHZ are generated.

The second method of fluorescence lifetime determination is by thephase-modulation method. Instead of using a pulsed light source ofexcitation, the method uses a light whose intensity is modulatedsinusoidally. The emission of the sample, therefore, follows the samesinusoidal variations. The modulated emission is delayed in phasebecause of the excited lifetime of the fluorescent molecules. Themagnitude of the phase shift (φ) is directly related to the lifetime ofthe fluorophore. Furthermore, there is demodulation of the emission. Inother words, the amplitude of the final emission is smaller in amplitudethan that of the excitation light. Both the phase angle (φ) and thedemodulation factor (m=BA/ba) are measured and used to calculate thephase (τ_(e)) and modulation lifetimes (τ_(m)) (Lakowicz, 1986).

$\tau_{p} = \frac{\tan \; \phi}{\omega}$$\tau_{m} = \frac{\sqrt{\left\lbrack {\left( {1/m^{2}} \right) - 1} \right.}}{\omega}$

In single exponential decay, τ_(p)=τ_(m)=τ, the actual fluorescencelifetime.

The basic theory behind phase modulation lifetime determination can beapplied to a two-dimensional imaging system. The following descriptionis based on from Gadella et al., 1993 and Lakowicz and Szmacinski, 1996,each of which are hereby incorporated by reference. The method uses again-modulated image intensifier that generates an image that has anintensity related to the phase shift of the emission signal. With use ofseveral phase-sensitive images collected with various electronic delaysor phase shifts, it is possible to calculate the lifetime image of theobject. To understand this further, it is necessary to present theequation that describes the time-averaged, phase-sensitive intensityfrom a certain position r:

I(r,η−D)=I _(o)(r)[I+½_(mD) m(r)cos(Θ(r)−Θ_(D))]

where r denotes the pixel position, Θ_(D) is the phase angle of the gainmodulation signal, Θ(r) is the phase of the emission, m_(D) is the gainmodulation of the detector, m(r) is the modulated amplitude of theemission, I_(o)(r) is the original intensity of the pixel (which dependson concentration). The equation describes the intensity of a given pixelas a function of two controlled parameters (Θ_(D) and m_(D)) and threeunknowns I_(o)(r), θ(r), m(r)). Recall that the lifetime (T) can bedetermined if either θ(r) or m(r) is known. Since there are threeunknowns, at least three different images are needed to determine thelifetime of the specimen. By controlling the phase angle of the gainmodulation signal (Θ_(D)), a series of phase-sensitive images aregenerated and hence lifetimes can be determined.

The apparatus for phase-modulation FLIM is described in Lakowicz andSzmacinski, 1996. Excitation is provided by the output of acavity-dumped laser, which is synchronously pumped by a mode-lockedNd:YAG laser. The excitation light is expanded by a laser beam expander.The gated image intensifier is positioned between the target and the CCDcamera. The gain of image intensifier is modulated using output of afrequency synthesizer. A CCD camera captures the phase sensitive images.A computer with FLIM software processes the output to generate alifetime image.

Other interactions involved in the method will produce a nuclearradiation signal. As a radiolabel on a polymer passes through thedefined region of detection, such as the station, nuclear radiation isemitted, some of which will pass through the defined region of radiationdetection. A detector of nuclear radiation is placed in proximity of thedefined region of radiation detection to capture emitted radiationsignals. Many methods of measuring nuclear radiation are known in theart including cloud and bubble chamber devices, constant current ionchambers, pulse counters, gas counters (i.e., Geiger-Muller counters),solid state detectors (surface barrier detectors, lithium-drifteddetectors, intrinsic germanium detectors), scintillation counters,Cerenkov detectors, etc.

Other types of signals generated are well known in the art and have manydetections means which are known to those of skill in the art. Amongthese include opposing electrodes, magnetic resonance, and piezoelectricscanning tips. Opposing nanoelectrodes can function by measurement ofcapacitance changes. Two opposing electrodes create an area of energystorage, which is effectively between the two electrodes. It is knownthat the capacitance of two opposing electrodes change when differentmaterials are placed between the electrodes. This value is known as adielectric constant. Changes in the dielectric constant can be measuredas a change in the voltage across the two electrodes. In the presentexample, different nucleotide bases or units of a polymer may give riseto different dielectric constants. The capacitance changes as thedielectric constant of the unit of the polymer per the equation:C=KC_(o), where K is the dielectric constant and C_(o) is thecapacitance in the absence of any bases. The voltage deflection of thenanoelectrodes is then outputted to a measuring device, recordingchanges in the signal with time.

A nanosized NMR detection device can be constructed to detect thepassage of specific spin-labeled polymer units. The nanosized NMRdetection device consists of magnets which can be swept and a means ofirradiating the polymer with electromagnetic energy of a constantfrequency (This is identical to holding the magnetic field constantwhile the electromagnetic frequency is swept). When the magnetic fieldreaches the correct strength, the nuclei absorb energy and resonanceoccurs. This absorption causes a tiny electric current to flow in anantenna coil surrounding the sample. The signal is amplified and outputto a recording device. For known labeled compounds, the time ofdetection is much faster than current means of NMR detection where afull spectra of the compound in question is required. Known labeledunits of polymers have known chemical shifts in particular regions,thereby eliminating the need to perform full spectral sweeps, loweringthe time of detection per base to micro or milliseconds.

A nanoscale piezoelectric scanning tip can be used to read the differentunits of the polymer based on physical contact of the different polymerunits with the tip. Depending on the size and shape of the polymer unit,different piezoelectric signals are generated, creating a series of unitdependent changes. Labels on units are physically different than nativeunits and can create a ready means for detection via a piezoelecticscanning tip. Upon contact of a polymer unit with the tip, thepiezoelectric crystals change and give rise to a current which isoutputted to a detection device. The amplitude and duration of thecurrent created by the interaction of the polymer unit and the tip ischaracteristic of the polymer unit.

Optical detectable signals are generated, detected and stored in adatabase the signals can be analyzed to determine structural informationabout the polymer. The computer may be the same computer used to collectdata about the polymers, or may be a separate computer dedicated to dataanalysis. A suitable computer system to implement the present inventiontypically includes an output device which displays information to auser, a main unit connected to the output device and an input devicewhich receives input from a user. The main unit generally includes aprocessor connected to a memory system via an interconnection mechanism.The input device and output device also are connected to the processorand memory system via the interconnection mechanism.

Nanochannels can be prepared by electroless deposition procedures whichproduce a metal fibril running the complete width of a polycarbonatetemplate membrane. The membrane can also be produced such that bothfaces of the membrane are covered with thin metal films to produce ananodisk electrode ensemble, one of the metal films can be removed toexpose the surface of the membrane. The metal films can be removed toexpose the surface of the membrane. These electrodes can be connected attheir bases to a common current collector. This assembly is useful forexamining changes in current as polymers flow through changes inconductance can be measured. The preparation of such plates is describedin Martin, C. P. R., Science, 266:1961-1965 (1994).

Computer programs for data analysis of the detected signals are readilyavailable from CCD manufacturers. Such programs may be executed using ageneral purpose computer such as described below. For the methods of theinvention, only operations on single pixels need to be performed (pointoperations). The complexity of the point operations depend on the methodof imaging used. Intensity based imaging offers the fastest manipulationof data and because only arithmetic is performed on the individualpixels. Regardless of the imaging technique (intensity, TGFM, or FLIM),the algorithms performed on each pixel in each method are consideredlow-level when compared to global, whole frame operations that need tobe performed in certain more complex imaging situations.

It should be understood that one or more output devices may be connectedto the computer system. Example output devices include a cathode raytube (CRT) display, liquid crystal displays (LCD), printers,communication devices such as a modem, and audio output. It should alsobe understood that one or more input devices may be connected to thecomputer system. Example input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication device, and data inputdevices such as sensors. It should be understood the invention is notlimited to the particular input or output devices used in combinationwith the computer system or to those described herein.

The computer system may be a general purpose computer system which isprogrammable using a high level computer programming language, such asAC @. The computer system may also be specially programmed, specialpurpose hardware. In a general purpose computer system, the processor istypically a commercially available processor, of which the series x86processors, available from Intel, and similar devices from AMD andCyrix, the 680×0 series microprocessors available from Motorola, thePowerPC microprocessor from IBM and the Alpha-series processors fromDigital Equipment Corporation are examples. Many other processors areavailable. Such a microprocessor executes a program called an operatingsystem, of which WindowsNT, UNIX, DOS, VMS and OS8 are examples, whichcontrols the execution of other computer programs and providesscheduling, debugging, input/output control, accounting, compilation,storage assignment, data management and memory management, andcommunication control and related services. The processor and operatingsystem define a computer platform for which application programs inhigh-level programming languages are written.

A memory system typically includes a computer readable and writeablenonvolatile recording medium, of which a magnetic disk, a flash memoryand tape are examples. The disk may be removable, known as a floppydisk, or permanent, known as a hard drive. A disk has a number of tracksin which signals are stored, typically in binary form, i.e., a forminterpreted as a sequence of one and zeros. Such signals may define anapplication program to be executed by the microprocessor, or informationstored on the disk to be processed by the application program.Typically, in operation, the processor causes data to be read from henonvolatile recording medium into an integrated circuit memory element,which is typically a volatile, random access memory such as a dynamicrandom access memory (DRAM) or static memory (SRAM). The integratedcircuit memory element allows for faster access to the information bythe processor than does the disk. The processor generally manipulatesthe data within the integrated circuit memory and then copies the datato the disk when processing is completed. A variety of mechanisms areknown for managing data movement between the disk and the integratedcircuit memory element, and the invention is not limited thereto. Itshould also be understood that the invention is not limited to aparticular memory system.

It should be understood the invention is not limited to a particularcomputer platform, particular processor, or particular high-levelprogramming language. Additionally, the computer system may be amultiprocessor computer system or may include multiple computersconnected over a computer network.

The data stored about the polymers may be stored in a database, or in adata file, in the memory system of the computer. The data for eachpolymer may be stored in the memory system so that it is accessible bythe processor independently of the data for other polymers, for exampleby assigning a unique identifier to each polymer.

The information contained in the data and how it is analyzed depends onthe number and type of labeled units that were caused to interact withthe agent to generate signals. For instance if every unit of a singlepolymer, each type of unit (e.g., all the A's of a nucleic acid) havinga specific type of label, is labeled then it will be possible todetermine from analysis of a single polymer the order of every unitwithin the polymer. If, however, only one of the four types of units ofa polymer such as a nucleic acid is labeled then more data will berequired to determine the complete sequence of the nucleic acid. Severallabeling schemes and methods for analyzing using the computer systemdata produced by those schemes are described in more detail below. Thelabeling strategies are described with respect to nucleic acids for easeof discussion. Each of these strategies, however, is useful for labelingall polymers.

Several different strategies of labeling are possible, involvingpermutations of different types of nucleotides labeled, differentpercentage of nucleotides labeled, and single-stranded ordouble-stranded labeling and diversity labels, such as compound whichbind to a polymer having a specific sequence (diversity labels arediscussed in more detail below relative to specific embodiments). Thesimplest labeling scheme involves the labeling of all four nucleotideswith different labels. Labeling schemes using three, two, or even onelabel are also possible.

A four nucleotide labeling scheme can be created where the A's, C's,G's, and T's of a target DNA is labeled with different labels. Such amolecule, upon traversing an interaction station, will generate a linearorder of signals which correspond to the linear sequence of nucleotideson the target DNA. The advantage of using a four nucleotide strategy isits ease of data interpretation and the fact that the entire sequence ofunits can be determined from a single labeled polymer. Adding extrinsiclabels to all four bases, however, may cause steric hindrance problems.In order to reduce this problem the intrinsic properties of some or allof the nucleotides may be used to label the nucleotides. As discussedabove, nucleotides are intrinsically labeled because each of the purinesand pyrimidines have distinct absorption spectra properties. In each ofthe labeling schemes described herein the nucleotides may be eitherextrinsically or intrinsically labeled but it is preferred that at leastsome of the nucleotides are intrinsically labeled when the fournucleotide labeling method is used. It is also preferred that whenextrinsic labels are used with the four nucleotide labeling scheme thatthe labels be small and neutral in charge to reduce steric hindrance.

A three nucleotide labeling scheme in which three of the fournucleotides are labeled may also be performed. When only three of thefour nucleotides are labeled analysis of the data generated by themethods of the invention is more complicated than when all fournucleotides are labeled. The data is more complicated because the numberand position of the nucleotides of the fourth unlabeled type must bedetermined separately. One method for determining the number andposition of the fourth nucleotide utilizes analysis of two differentsets of labeled nucleic acid molecules. For instance, one nucleic acidmolecule may be labeled with A, C, and G, and another with C, G, and T.Analysis of the linear order of labeled nucleotides from the two setsyields sequence data. The three nucleotides chosen for each set can havemany different possibilities as long as the two sets contain all fourlabeled nucleotides. For example, the set ACG can be paired with a setof labeled CGT, ACT or AGT.

The sequence including the fourth nucleotide also may be determined byusing only a single labeled polymer rather then a set of at least twodifferently labeled polymers by identifying the position of the fourthnucleotide on the polymer. This can be accomplished by determining thedistance between labeled nucleotides on a nucleic acid molecule. Forexample A, C, and G are labeled and the detectable signals generatedindicated that the nucleic acid molecule had a sequence of AGGCAAACG(SEQ. ID. No. 2). If the distances between each of the nucleotides inthe nucleic acid molecule are equivalent to the known inter-nucleotidedistance for a particular combination of nucleotides except the distancebetween G and G is twice the normal inter-nucleotide distance then a Tis positioned between the two G's and the entire molecule has a sequenceof AGTGCAAACG (SEQ. ID. No. 3). The distance between nucleotides can bedetermined in several ways. Firstly, the polymer and the agent may bemoved relative to one another in a linear manner and at a constant rateof speed such that a single unit of the nucleic acid molecule will passthe agent at a single time interval. If two time intervals elapsebetween detectable signals then the unlabeled nucleotide which is notcapable of producing a detectable signal is present within thatposition. This method of determining the distance between bases isdiscussed in more detail below in reference to random one base labeling.Alternatively the polymer and the agent may be caused to interact withone another such that each labeled unit interacts simultaneously with anagent to produce simultaneous detectable signals. Each detectable signalgenerated occurs at the point along the polymer where the unit ispositioned. The distance between the detectable signals can becalculated directly to determine whether an unlabeled unit is positionedanywhere along the nucleic acid molecule.

Nucleic acid molecules may also be labeled according to a two nucleotidelabeling scheme. Six sets of two nucleotide labeled nucleic acidmolecule can be used to resolve the data and interpret the nucleotidesequence. Ambrose et al., 1993 and Harding and Keller, 1992 havedemonstrated the synthesis of large fluorescent DNA molecules with twoof the nucleotides completely extrinsically labeled. The average size ofthe molecules were 7 kb. Six different combinations of two nucleotidelabeling are possible using the following formula:

${1.\mspace{14mu} \left( {{}_{}^{}{}_{}^{}} \right)} = {\frac{n\; t}{{{kt}\left( {n - k} \right)}t} = {\frac{4}{2{t2t}} = 6}}$

where n nucleotides are taken k at a time. The possible combinations areAC, AG, AT, CG, CT, and GT. Knowledge of the linear order of the labelsin each of the sets allows for successful reconstruction of the nucleicacid sequence. Using a 4-mer (5′ ACGT 3′) as a model sequence, thetheory can be demonstrated. The first set, AC, gives the informationthat there must be a C after the A. This does not give information aboutthe number of nucleotides intervening the A and the C nor does it giveinformation about any G's or T's preceding the A. The second set, AG,shows that there is also a G after the A. Set AT shows there is a Tafter the A. From these three sets, it is then known that the target DNAis a 4-mer and that one C, one G, and one T follow the A. The subsequentsets give information on the ordering of these three nucleotidesfollowing the A. Set CG shows that G follows C. Set CT shows that Tfollows C. Set GT finishes the arrangement to give the final decipheredsequence of 5′ ACGT 3′. In addition to the method using six labeled setsof nucleic acid molecules, the sequence can be established by combinginformation about the distance between labeled nucleotides generatingdetectable signals as described above and information obtained fromfewer than six sets of two nucleotide labeled nucleic acid molecules.

A fourth labeling scheme, the random one nucleotide labeling scheme alsomay be used. In this method, distance information which is obtained byeither population analysis and/or instantaneous rate of DNA movement isused to determine the number of nucleotides separating two labelednucleotides. Analysis of four differently labeled target moleculesyields the complete sequence.

One method of analysis with these labeling methods includes the use ofcomplementary base information. FIG. 1 demonstrates the labelingstrategy in which two differently labeled DNA samples are required. Thefirst sample has two of its non-complementary bases randomly labeledwith the same fluorophore. Non-complementary pairs of bases are AC, AG,TC, and TG. The second sample has one of its bases randomly labeled. Thebase chosen for the second sample can be any of the four bases. In theexample given, the two non-complementary bases are chosen to be A and C.As a result, two samples are prepared, one with labeled A's and C's andanother with labeled A's. The DNA is genomically digested, end-labeled,purified, and analyzed by nanochannel FRET sequencing. Thesequence-specific FRET information arising from each fragment is sortedinto one of two complementary strand groups. Sorting allows populationanalysis to determine the positions of all the desired bases. The figureillustrates the generation of sequence information from the sorted data.The first group of analyzed information yields the positions of all theA's and C's on one strand. The second group analyzed yields knowledge ofall the A's and C's on one strand. The same procedure is applied to thecomplementary stand. Knowledge of the complementary strand's A's and C'sis identical to knowledge of the T's and G's on the other stand. Theresult is sequence reconstruction. To cross-verify the sequence, theprocess can be repeated for the other pairs of non-complementary basessuch as TG, TC and AG.

There are two methods of determining the distance between bases. Onerequires determining the instantaneous rate of DNA movement, which isreadily calculated from the duration of energy transfer or quenching fora particular label. Another involves analyzing a population of targetDNA molecules and its corresponding Gaussian distance distributions.

The instantaneous rate method, involves a determination of distanceseparation based on the known instantaneous rate of DNA movement (v)multiplied by the time of separation between signals (t). Instantaneousrate is found by measuring the time that it takes for a labelednucleotide to pass by the interaction station. Since the length of theconcentrated area of agent (d) is known (through calibration andphysical measurement of the localized region of the agent, e.g., thethickness of a concentrated donor fluorophore area), the rate is simplyv=d/t. As shown in FIG. 2 analysis of raw data demonstrating changes inenergy emission patterns resulting from sequential detectable signalswhen plotted produces a curve which from left to right shows two energyintensity decreases, followed by two energy intensity increases. Theplateau from the first energy intensity decrease (denoted t₁) is doublethat of the second plateau (t₂). The length of the interaction stationis given as 51 Å. From this given information, the number of labelednucleotides is known. Furthermore, the distance of separation of the twois determined by relating the rate of DNA movement to the time of thedonor intensity plateaus. The number of labeled nucleotides is simplydenoted by the number of intensity decreases. In FIG. 2, there are twointensity decreases. Accordingly, there must be two detectable labels onthe DNA. To determine the distance of base separation, it is necessaryto know the instantaneous rate of DNA movement, which is found byknowing the time for one labeled nucleotide to cross the localizedregion of the agent and the length of the localized region of the agent.The length of the localized region of the agent is given as 51 {acuteover (Å)}. The time for one labeled nucleotides crossing the localizedregion of the agent is bounded by the first intensity decrease and thefirst intensity increase (denoted as the gray shaded region, 7.5 s). Therate of DNA movement is 6.8 Å/s. The base separation is derived from thetime separating the labeled nucleotides (t₁=5 s) multiplied by the rate(6.8 {acute over (Å)}), which is equal to 10 base pairs. As a means ofcross-verification, 51 Å−t₂v also yields the base separation.

In the population method the entire population of labeled nucleotide isconsidered. Knowledge of the length of the localized region of the agentand instantaneous rate, as required for the rate method, is notnecessary. Use of population analyses statistically eliminates the needfor precision measurements on individual nucleic acid molecules.

An example of population analyses using five nucleic acid molecules eachtraversing a nanochannel is described below. Five molecules representinga population of identical DNA fragments are prepared. In a constantelectric field, the time of detection between the first and secondlabeled nucleotide should be identical for all the DNA molecules. Underexperimental conditions, these times differ slightly, leading to aGaussian distribution of times. The peak of the Gaussian distribution ischaracteristic of the distance of separation (d) between two labelednucleotides.

An additional example utilizing a population of one nucleotide randomlylabeled nucleic acid molecule (six molecules represent the population)further illustrates the concept of population analysis and thedetermination of distance information. The nucleic acid is end-labeledto provide a reference point. With enough nucleic acid molecules, thedistance between any two A's can be determined. Two molecules, whenconsidered as a sub-population, convey the base separation molecules,distributions of 4 and 6 base separations are created. Extending thesame logic to rest of the population, the positions of all the A's onthe DNA can be determined. The entire sequence is generated by repeatingthe process for the other three bases (C, G, and T).

In addition to labeling all of one type of unit, it is possible to use aone-nucleotide labeling scheme where not every nucleotide of one type islabeled. An outline of this method is shown in FIG. 3 which shows arepresentative population of random A-labeled fragments for a 16-merwith the sequence 5′ ACGTACGTACGTACGT 3′ (SEQ. ID. No. 1). Eachindividually labeled DNA molecule has half of its A's labeled inaddition to 5′ and 3′ end labels. With a large population of randomlylabeled fragments, the distance between every successive A on the targetDNA can be found. The end labels serve to identify the distance betweenthe ends of the DNA and the first A. Repeating the same analysis for theother nucleotides generates the sequence of the 16-mer. The advantagesof using such a method includes lack of steric effects and ease oflabeling. This type of labeling is referred to as random labeling. Apolymer which is “randomly labeled” is one in which fewer than all of aparticular type of unit are labeled. It is unknown which units of aparticular type of a randomly labeled polymer are labeled.

As mentioned briefly above, various combinations of the labeling schemescan be used together. In all of the methods listed above, either orderedlinear information from signals or distance information betweennucleotides is considered. These two aspects can be combined to givemethods that rely on both ordered linear and distance information. Forexample a random one nucleotide labeling strategy expanded to a randomfour nucleotide labeling strategy. Random four nucleotide labeling iswhere a fraction of all four nucleotides is labeled. A population ofmolecules have a fraction of the four nucleotides labeled. Each of thefour nucleotides have a unique label. Analysis of a randomly labeledpopulation results in generation of the sequence data.

The use of double-stranded DNA allows for variations. A single-strandedtwo-nucleotide labeling scheme can be performed when two of thenucleotides on one strand of DNA are fully replaced by labelednucleotides. To reduce the steric constraints imposed by twoextrinsically labeled nucleotides while preserving the theory behindtwo-nucleotide labeling, it is possible to label one nucleotide fully oneach of the complementary strands to achieve the same end. This methodinvolves using double-stranded DNA in which each strand is labeled witha different label. Six differently labeled duplex DNA sets will producea data set which is adequate to provide sequence information. Eachcomplementary strand of DNA should have one of the nucleotides labeled.In each of the duplex DNA sets, the equivalent of two differentnucleotides (possible combinations are AC, AG, AT, CG, CT, GT) arelabeled. When both complementary strands have the adenines labeled, thisis equivalent to the combination AT. In duplex two-nucleotide labeling,the advantage is that only one nucleotide on each strand is labeled,allowing longer labeled strands to be synthesized as compared totwo-nucleotide labeling on single-stranded DNA. In practice, it has beenshown that synthesis of DNA fragments with one nucleotide completelylabeled can be achieved with lengths much greater than 10 kb (Ambrose etal., 1993; Harding and Keller, 1992).

One use for the methods of the invention is to determine the sequence ofunits within a polymer. Identifying the sequence of units of a polymer,such as a nucleic acid, is an important step in understanding thefunction of the polymer and determining the role of the polymer in aphysiological environment such as a cell or tissue. The sequencingmethods currently in use are slow and cumbersome. The methods of theinvention are much quicker and generate significantly more sequence datain a very short period of time.

Sequencing of a polymer may encompass the sequencing of the entirepolymer or portions of the polymer or even the identification of anindividual unit on the polymer. One method for identifying an individualunit of a polymer involves the steps of transiently exposing theindividual unit of the polymer, the identity of which is unknown, to anagent selected from the group consisting of electromagnetic radiation, aquenching source and a fluorescence excitation source to generate aninteraction with a detectable signal characteristic of the individualunit, and detecting and distinguishing the signal from signals generatedfrom adjacent signal generating units of the polymer as an indication ofthe identity of the individual unit.

The individual unit is “transiently exposed” to the agent in order toproduce a detectable signal characteristic of the individual unit.“Transiently exposed” as used herein means that the unit is positionedwithin interactive proximity of the agent for enough time to produce asignal and then is moved out of interactive proximity. The exact lengthof time required to produce a signal will depend on the individual unitand the agent involved but generally the amount of time is between onenanosecond and one second.

The signal characteristic of the individual unit is distinguished fromsignals generated from adjacent signal generating units of the polymer.An “adjacent signal generating unit” is the unit nearest to theindividual unit which when exposed to the agent produces a detectablesignal. It is not necessarily the unit which is directly linked to theindividual unit unless the unit which is directly linked is labeled(intrinsically or extrinsically) and produces a detectable signal.

In the case when the agent is one or more fluorophores the interactiveproximity between the agent and the unit is the energy transferproximity and the signal produced is fluorescence resonance energytransfer. “Energy transfer proximity” as used herein is the distancebetween the unit and the fluorophore which allows interaction betweentwo complementary sources if one source overlaps with the absorptionspectrum of the other source. “Fluorescence resonance energy transfer”as used herein is the transfer of photonic energy between fluorophoreswith overlapping emission and absorption spectra.

Another method for identifying an individual unit of a polymer involvesthe steps of transiently moving the individual unit of the polymerrelative to a station, the identity of the individual unit beingunknown, detecting a signal arising from a detectable physical change inthe unit or the station, and distinguishing said signal from signalsarising from exposure of adjacent signal generating units of the polymerto the station as an indication of the identity of the individual unit.

Thus in one aspect, the methods of the invention can be used to identifyone, some, or all of the units of the polymer. This is achieved byidentifying the type of individual unit and its position on the backboneof the polymer by determining whether a signal detected at thatparticular position on the backbone is characteristic of the presence ofa particular labeled unit.

The methods of the invention also are useful for identifying otherstructural properties of polymers. The structural information obtainedby analyzing a polymer according to the methods of the invention mayinclude the identification of characteristic properties of the polymerwhich (in turn) allows, for example, for the identification of thepresence of a polymer in a sample or a determination of the relatednessof polymers, identification of the size of the polymer, identificationof the proximity or distance between two or more individual units of apolymer, identification of the order of two or more individual unitswithin a polymer, and/or identification of the general composition ofthe units of the polymer. Such characteristics are useful for a varietyof purposes such as determining the presence or absence of a particularpolymer in a sample. For instance when the polymer is a nucleic acid themethods of the invention may be used to determine whether a particulargenetic sequence is expressed in a cell or tissue. The presence orabsence of a particular sequence can be established by determiningwhether any polymers within the sample express a characteristic patternof individual units which is only found in the polymer of interest i.e.,by comparing the detected signals to a known pattern of signalscharacteristic of a known polymer to determine the relatedness of thepolymer being analyzed to the known polymer. The entire sequence of thepolymer of interest does not need to be determined in order to establishthe presence or absence of the polymer in the sample. Similarly themethods may be useful for comparing the signals detected from onepolymer to a pattern of signals from another polymer to determine therelatedness of the two polymers.

The proximity of or distance between two individual units of a polymermay be determined according to the methods of the invention. It isimportant to be able to determine the proximity of or distance betweentwo units for several reasons. Each unit of a polymer has a specificposition along the backbone. The sequence of units serves as a blueprintfor a known polymer. The distance between two or more units on anunknown polymer can be compared to the blueprint of a known polymer todetermine whether they are related. Additionally the ability todetermine the distance between two units is important for determininghow many units, if any, are between the two units of interest.

In order to determine the proximity of two individual units of a polymerof linked units the polymer is moved relative to a station, where eachindividual unit is exposed to the station to produce a characteristicsignal arising from a detectable physical change in the unit or station.Each of the characteristic signals generated is then detected and theamount of time elapsed between detecting each characteristic signal ismeasured as described above. The amount of time elapsed is indicative ofthe proximity of the two individual units. The station may be aninteraction station and the unit may be exposed to an agent to producean electromagnetic signal.

A “signal characteristic of an interaction” as used herein is a signalwhich is expected to result from the interaction of the station and aspecific labeled unit. The specific signal generated will depend on thetype of station as well as the type of labeled unit. For instance if thestation is an agent which is electromagnetic radiation and the labeledunit is a fluorophore then the interaction between the two will resultin the emission of electromagnetic radiation by the fluorophore at awavelength at which the fluorophore is known to emit. If the station isan agent which is a scintillation layer and the unit is radioactive thenthe interaction between the two will result in the emission ofelectromagnetic radiation in the form of fluorescence.

It is possible to determine the order of the units of a polymer usingthe methods of the invention. In one aspect of the invention the orderof two individual units of a polymer can be determined by moving thepolymer linearly with respect to a station and exposing two of theindividual units to the station to produce first and second detectablesignals arising from physical changes in the station or the unit. Theorder of the signals is an indication of the order of the two individualunits.

The general composition of the units of the polymer may also bedetermined by the methods of the invention. For instance, if the polymeris a nucleic acid the methods of the invention can provide informationon the percentage of purines vs. pyrimidines or the percentage of A, C,T, and G in the nucleic acid.

Quantitative information on the size of the sample may also bedetermined by the methods of the invention. For instance, the size of apolymer can be determined by determining the number of individual unitswhich make up the polymer. The number of units which make up the polymeris determined by measuring the amount of time that is required for theentire polymer to traverse past an agent at an interaction site in alinear manner and dividing that by the average length of time for anindividual unit of that particular type of polymer to completelytraverse past the site.

In addition to information about a specific unit the methods of theinvention may be used to identify greater than one unit at a time inorder to provide information about a polymer. In one aspect the methodis carried out by providing a labeled polymer of linked units, detectingsignals from labeled unit specific markers of less than all of thelinked units, and storing a signature of the signals detected to analyzethe polymer. In this aspect of the invention each unit of the labeledpolymer may be labeled with a unit specific marker or less than all ofthe units may be labeled with a unit specific marker.

This method is particularly useful for analyzing multiple units of apolymer at one time. This is accomplished by using a unit specificmarker which is labeled and which interacts with more than one unit in asequence specific manner. As used herein a “unit specific marker” is acompound which specifically interacts with one or more units of apolymer and is capable of identifying those units. For instance a unitspecific marker for a nucleic acid molecule can be a labeled dimers,trimers, etc. which bind to a specific sequence of bases, such as TG,AG, ATC, etc. By identifying the presence or position of the labeledmarkers structural information about the polymer can be derived. Forinstance, the presence of the marker on a polymer can reveal theidentity of the polymer. This enables the presence or absence of apolymer in a solution or mixture of polymers to be determined. Theorder, distance, number etc. of the markers on a polymer can provideinformation about the sequence or composition of a polymer. Other unitspecific markers include but are not limited to sequence specific majorand minor groove binders and intercallators, sequence specific DNA andpeptide binding proteins, sequence specific peptide-nucleic acids, etc.Many such unit specific markers exist and are well known to those ofskill in the art.

This type of analysis can be used in one embodiment to identify DNAfragments by analyzing the hybridization patterns of multiple probes toindividual fragments of polymers. The current state-of-the-art methodsfor hybridization analysis of DNA rely upon DNA chips. The methods ofthe invention is advantageous for a number of reasons. The number, type,order, and distance between the multiple probes bound to an unknownfragment of DNA can be determined. This information can be used toidentify the number of differentially expressed genes unambiguously.Current hybridization approaches can only determine the type of probesbound to a given fragment. Furthermore, the methods of the invention areable to quantitate precisely the actual number of particular expressedgenes. Current methods rely on quantitation of fluorescence intensities,which often give rise to errors due to non-linearities in the detectionsystem. Given the great amount of information generated, the methods ofthe invention do not require a selection of expressed genes or unknownnucleic acids to be assayed. This is in contrast to the requirement ofdifferent DNA chips for different genes, sets of expressed genes to beanalyzed, and also different organisms. The methods of the invention canidentify the unknown expressed genes by computer analysis of thehybridization patterns generated. The data obtained from linear analysisof the DNA probes are then matched with information in a database todetermine the identity of the target DNA. The methods can thus analyzeinformation from hybridization reactions, which can then be applied todiagnostics and determination of gene expression patterns.

A “signature” as used herein is a sequence-specific signal arising froma labeled polymer. The signature includes information about thestructure of the polymer. For instance, the signature of a polymer maybe defined by a series of consecutive units or by specific units spaceda particular distance apart from one another. The signature of thepolymer identifies the polymer. Signatures are useful for uniquelyidentifying fragments by identifying bases at certain positions alongthe length of a strand of DNA. The probability of knowing any oneposition is ¼. Unambiguous identification of a fragment comes withroughly twenty positions identified (¼²⁰=9.1×120⁻¹³). For a fragmentwith 20 detected labels and 10% detection/labeling, the size of thefragment needs to be only 200 base pairs. The proposed read length is onthe order of kilobases, which should unambiguously identify anyfragment. The identification of fragments allows for grouping by similarsequences, making sequence reconstruction by population analysispossible.

A preferred method of analysis of the invention involves the use ofradioactively labeled polymers. The type of radioactive emissioninfluences the type of detection device used. In general, there arethree different types of nuclear emission including alpha, beta, andgamma radiation. Alpha emission cause extensive ionization in matter andpermit individual counting by ionization chambers and proportionalcounters, but more interestingly, alpha emission interacting with mattermay also cause molecular excitation, which can result in fluorescence.The fluorescence is referred to as scintillation. Beta decay which isweaker than alpha decay can be amplified to generate an adequate signal.Gamma radiation arises from internal conversion of excitation energy.Scintillation counting of gamma rays is efficient and produces a strongsignal. Sodium iodide crystals fluoresce with incident gamma radiation.

A “scintillation” layer or material as used herein is any type ofmaterial which fluoresces or emits light in response to excitation bynuclear radiation. Scintillation materials are well known in the art.Aromatic hydrocarbons which have resonance structures are excellentscintillators. Anthracene and stilbene fall into the category of suchcompounds. Inorganic crystals are also known to fluoresce. In order forthese compounds to luminesce, the inorganic crystals must have smallamounts of impurities, which create energy levels between valence andconduction bands. Excitation and de-excitation can therefore occur. Inmany cases, the de-excitation can occur through phosphorescent photonemission, leading to a long lifetime of detection. Some commonscintillators include NaI (T1), ZnS (Ag), anthracene, stilbene, andplastic phosphors.

Many methods of measuring nuclear radiation are known in the art andinclude devices such as cloud and bubble chamber devices, constantcurrent ion chambers, pulse counters, gas counters (i.e., Geiger-Mullercounters), solid state detectors (surface barrier detectors,lithium-drifted detectors, intrinsic germanium detectors), scintillationcounters, Cerenkov detectors, etc.

Analysis of the radiolabeled polymers is identical to other means ofgenerating polymer dependent impulses. For example, a sample withradiolabeled A's can be analyzed by the system to determine relativespacing of A's on a sample DNA. The time between detection of radiationsignals is characteristic of the polymer analyzed. Analysis of fourpopulations of labeled DNA (A's, C's, G's, T's) can yield the sequenceof the polymer analyzed. The sequence of DNA can also be analyzed with amore complex scheme including analysis of a combination of dual labeledDNA and singly labeled DNA. Analysis of a A and C labeled fragmentfollowed by analysis of a A labeled version of the same fragment yieldsknowledge of the positions of the A's and C's. The sequence is known ifthe procedure is repeated for the complementary strand. The system canfurther be used for analysis of polymer (polypeptide, RNA,carbohydrates, etc.), size, concentration, type, identity, presence,sequence and number.

The methods described above can be performed on a single polymer or onmore than one polymer in order to determine structural information aboutthe polymer. The invention also encompasses the practice of the methodsdescribed above on multiple polymers. These methods and an apparatus forperforming the methods of the invention simultaneously on a plurality ofpolymers are described in detail below.

Some of the methods described above are based on an interactioninvolving energy transfer or quenching to produce a detectable signal.The involvement of energy transfer or quenching is described in eitherone of two limitations in these embodiments of the invention. Onelimitation is that the agent involved in the interaction is selectedfrom the group consisting of electromagnetic radiation, a quenchingsource or a fluorescence excitation source. The other limitation is thatthe detectable signal is an electromagnetic radiation signal. It shouldbe apparent to one of ordinary skill in the art that each of the methodscan encompass the other limitation instead of the one described andstill encompass the notion of interaction involving energy transfer orquenching. For instance in addition to encompassing the method foranalyzing a polymer by exposing the units of the polymer to the agentselected from the group consisting of electromagnetic radiation, aquenching source, and a fluorescence excitation source to produce adetectable signal, the invention in these embodiments also encompasses amethod for analyzing a polymer by exposing the units of a polymer to anagent to produce a detectable electromagnetic signal.

In addition to the methods involving energy transfer, quenching orelectromagnetic radiation signals, the methods of the inventiondescribed above can be performed by detecting signals which arise from adetectable physical change in the unit of the polymer or the station. Asused herein a “detectable physical change” in the unit of the polymer orthe station is any type of change which occurs in the unit of thepolymer or the station as a result of exposing the unit to the station.Once the unit is exposed to the station a detectable signal is created.The station may be for instance, an interaction station or a signalgeneration station, each of which is discussed in detail herein. Thetype of change that occurs in the station or the unit to produce thedetectable signal depends on the type of station and the type of unit.Several examples of station-unit combinations which undergo a change toproduce a detectable signal are discussed herein for exemplary purposes.Those of skill in the art will be able to derive other station-unitcombinations that fall within the scope of the invention.

When the interaction between the station and the unit of the polymer isbased on energy transfer, either the unit or the station or both mayphysically change to produce a signal. In one embodiment the station maytransfer energy to the unit causing the unit to emit an energy unitspecific signal. The physical change which occurs in the unit resultsfrom the change in energy state. In another embodiment the unit maytransfer energy to the station causing the station to emit aunit-specific signal resulting from the specific energy transfer. Instill other embodiments a partner compound may cause the physical changewhich produces a signal. When the interaction occurs between aradioactive unit and a station the unit physically changes by releasingenergy.

Another aspect of the invention encompasses methods for analyzing aplurality of polymers. Each of the polymers is analyzed by sequentiallydetecting interactions of units of a plurality of polymers at a signalgeneration station. These methods include but are not limited to amethod for characterizing a test polymer, a method for sequencing apolymer, a method for determining the order of units of a polymer, amethod for determining the distance between units of a polymer, andanalyzing a set of polymers. These methods encompass but are not limitedto interactions resulting from energy transfer or quenching.

A method for characterizing a test polymer is performed by obtainingpolymer dependent impulses for each of a plurality of polymers,comparing the polymer dependent impulses of the plurality of polymers,determining the relatedness of the polymers based upon similaritiesbetween the polymer dependent impulses of the polymers, andcharacterizing the test polymer based upon the polymer dependentimpulses of related polymers.

A “polymer dependent impulse” as used herein is a detectable physicalquantity which transmits or conveys information about the structuralcharacteristics of only a single unit of a polymer. The physicalquantity may be in any form which is capable of being detected. Forinstance the physical quantity may be electromagnetic radiation,chemical conductance, electrical conductance, etc. The polymer dependentimpulse may arise from energy transfer, quenching, changes inconductance, mechanical changes, resistance changes, or any otherphysical changes. Although the polymer dependent impulse is specific fora particular unit, a polymer having more than one of a particularlabeled unit will have more than one identical polymer dependentimpulse. Additionally, each unit of a specific type may give rise todifferent polymer dependent impulses if they have different labels.

The method used for detecting the polymer dependent impulse depends onthe type of physical quantity generated. For instance if the physicalquantity is electromagnetic radiation then the polymer dependent impulseis optically detected. An “optically detectable” polymer dependentimpulse as used herein is a light based signal in the form ofelectromagnetic radiation which can be detected by light detectingimaging systems. When the physical quantity is chemical conductance thenthe polymer dependent impulse is chemically detected. A “chemicallydetected” polymer dependent impulse is a signal in the form of a changein chemical concentration or charge such as an ion conductance which canbe detected by standard means for measuring chemical conductance. If thephysical quantity is an electrical signal then the polymer dependentimpulse is in the form of a change in resistance or capacitance.

As used herein the “relatedness of polymers” can be determined byidentifying a characteristic pattern of a polymer which is unique tothat polymer. For instance if the polymer is a nucleic acid thenvirtually any sequence of 10 contiguous nucleotides within the polymerwould be a unique characteristic of that nucleic acid molecule. Anyother nucleic acid molecule which displayed an identical sequence of 10nucleotides would be a related polymer.

A “plurality of polymers” is at least two polymers. Preferably aplurality of polymers is at least 50 polymers and more preferably atleast 100 polymers.

The polymer dependent impulses may provide any type of structuralinformation about the polymer. For instance these signals may providethe entire or portions of the entire sequence of the polymer, the orderof polymer dependent impulses, or the time of separation between polymerdependent impulses as an indication of the distance between the units.

The polymer dependent impulses are obtained by interaction which occursbetween the unit of the polymer and the environment at a signalgeneration station. A “signal generation station” as used herein is astation that is an area where the unit interacts with the environment togenerate a polymer dependent impulse. In some aspects of the inventionthe polymer dependent impulse results from contact in a defined areawith an agent selected from the group consisting of electromagneticradiation, a quenching source, and a fluorescence excitation sourcewhich can interact with the unit to produce a detectable signal. Inother aspects the polymer dependent impulse results from contact in adefined area with a chemical environment which is capable of undergoingspecific changes in conductance in response to an interaction with amolecule. As a molecule with a specific structure interacts with thechemical environment a change in conductance occurs. The change which isspecific for the particular structure may be a temporal change, e.g.,the length of time required for the conductance to change may beindicative that the interaction involves a specific structure or aphysical change. For instance, the change in intensity of theinteraction may be indicative of an interaction with a specificstructure. In other aspects the polymer dependent impulse results fromchanges in capacitance or resistance caused by the movement of the unitbetween microelectrodes or nanoelectrodes positioned adjacent to thepolymer unit. For instance the signal generation station may includemicroelectrodes or nanoelectrodes positioned on opposite sides of thepolymer unit. The changes in resistance or conductance which occur as aresult of the movement of the unit past the electrodes will be specificfor the particular unit.

A method for determining the distance between two individual units isalso encompassed by the invention. In order to determine the distancebetween two individual units of a polymer of linked units the polymer iscaused to pass linearly relative to an signal generation station and apolymer dependent impulse which is generated as each of the twoindividual units passes by the signal generation station is measured.Each of the steps is then repeated for a plurality of similar polymers.A polymer is said to pass linearly relative to a signal generationstation when each unit of the polymer passes sequentially by the signalgeneration station.

Each of the steps is repeated for a plurality of similar polymers toproduce a data set. The distance between the two individual units canthen be determined based upon the information obtained from theplurality of similar polymers by analyzing the data set.

The method also includes a method for identifying a quantity of polymersincluding a label. For instance, it is possible to determine the numberof polymers having a specific unit or combination of units in a sample.In a sample of mRNA, for example, the number of a particular mRNApresent in the sample can be determined. This is accomplished byidentifying a pattern or signature characteristic of the desired mRNAmolecule. The sample of RNA can then be analyzed according to themethods of the invention and the number of mRNA molecules having thespecific pattern or signature can be determined.

As used herein “similar polymers” are polymers which have at least oneoverlapping region. Similar polymers may be a homogeneous population ofpolymers or a heterogenous population of polymers. A “homogeneouspopulation” of polymers as used herein is a group of identical polymers.A “heterogenous population” of similar polymers is a group of similarpolymers which are not identical but which include at least oneoverlapping region of identical units. An overlapping region typicallyconsists of at least 10 contiguous nucleotides. In some cases anoverlapping region consists of at least 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or 22 contiguous nucleotides.

A “plurality of similar polymers” is two or more similar polymers.Preferably a plurality of similar polymers is 50 or more similarpolymers. More preferably a plurality of similar polymers is 100 or moresimilar polymers.

A “data set” as used herein is a set of information defining the polymerdependent impulses generated by similar polymers. The data set isanalyzed as discussed above and the method of analysis used depends onthe type of labeling scheme used to generate the labeled polymers.Nucleic acid sequencing is a particularly preferred embodiment of themethods of the invention.

Currently, less than 5% of the human genome has been sequenced. Thistranslates into a small fraction of the ideal in human sequenceknowledge, which is the sequence of all individuals. For an instance,for the human population, there are 1.4×10¹⁹ (5 billion people×3×10⁹bases/person). So far, only 2×10⁻¹° percent of all human geneticinformation is known. The rate of sequencing of the human genome by allworld-wide efforts is roughly 3×10⁹/15 years, or 550,000 bases/day, at acost of >$1/base. Sequencing by the methods of the invention describedherein will constitute an inordinate breakthrough in the rate ofsequencing. The predicted time to complete one human genome with onemachine is ˜15 hours. Several dynamic arrays in parallel will be able tocomplete the sequence of one human genome in a fraction of an hour.

A method for sequencing a polymer of linked units is also encompassed bythe invention. The method is performed by obtaining polymer dependentimpulses from each of a plurality of overlapping polymers, at least aportion of each of the polymers having a sequence of linked unitsidentical to the other of the polymers, and comparing the polymerdependent impulses to obtain a sequence of linked units which isidentical in the plurality of polymers.

The plurality of overlapping polymers is a set of polymers in which eachpolymer has at least a portion of its sequence of linked units which isidentical to the other polymers. The portion of sequence which isidentical is referred to as the overlapping region and which includes atleast ten contiguous units.

In another aspect of the invention the order of units of a polymer oflinked units can be determined by moving the polymer linearly relativeto a signal generation station and measuring a polymer dependent impulsegenerated as each of two individual units, each giving rise to acharacteristic polymer dependent impulse pass by the signal generationstation. These steps are repeated for a plurality of similar polymersand the order of at least the two individual units is determined basedupon the information obtained from the plurality of similar polymers.

A method for analyzing a set of polymers, in which each of the polymersof the set is an individual polymer of linked units, is encompassed bythe invention. The method involves the step of orienting the set ofpolymers parallel to one another, and detecting a polymer specificfeature of the polymers.

The set of polymers are oriented parallel to one another. The polymersmay be oriented by any means which is capable of causing the polymers tobe positioned parallel to one another. For instance an electric fieldmay be applied to the polymers to cause them to be oriented in aparallel form. Preferably the orientation step is in a solution free ofgel.

A “polymer specific feature” as used herein is any structural feature ofpolymer which relates to its sequence. For instance a polymer specificfeature includes but is not limited to information about the polymersuch as the length of the polymer, the order of linked units in thepolymer, the distance between units of the polymer, the proximity ofunits in the polymer, the sequence of one, some or all of the units ofthe polymer, and the presence of the polymer.

The step of detecting the polymer specific feature may be performedsimultaneously for all of the polymers. This step encompasses thesequential detection of each of the units of all of the polymers. Thiscan be accomplished by passing linearly each of the polymers relative toa plurality of signal generation stations, and detecting anddistinguishing polymer dependent impulses generated as said polymerspass said signal generation stations.

The invention also includes a method for analyzing a set of polymers,each polymer of the set being an individual polymer of linked units. Themethod is performed by orienting the set of polymers in an electricfield, simultaneously moving the set of polymers through definedrespective channels, and detecting a polymer specific feature as thepolymers are moved through the channels. The step of simultaneouslymoving the set of polymers through respective channels is carried out bymoving one polymer per channel such that each unit passes the stationindividually. More than one polymer may be in the channel at a time ifthe polymers are positioned in tandem and only one unit interacts withone station at a time.

A “defined respective channel” as used herein is a channel in which thestructure is determined before the polymer enters the channel such thatthe polymer will follow a defined path as it passes through the channel.Channels such as those found in a gel matrix are not defined respectivechannels.

The methods of the invention may also be used to detect resonance energytransfer or quenching between two interactive partners capable of suchtransfer or quenching. As used herein resonance energy transfer (RET) isthe transfer of photonic energy between two compounds with overlappingemission and absorption spectra. Fluorescence resonance energy transfer(FRET) is the transfer of photonic energy between fluorophores. The twointeractive partners are any compounds which are capable of energytransfer or quenching i.e., light emissive compounds or quenchers.

The method is performed by bringing the two partners in close enoughproximity to permit such transfer or quenching, applying an agent to oneof said partners, the agent selected from the group consisting ofelectromagnetic radiation, a quenching source and a fluorescenceexcitation source, shielding fluorescence resonance energy transfer andquenching occurring from electromagnetic radiation emission andinteraction between the partners with a material shield, and detectingthe emitted electromagnetic radiation.

As used herein a “material shield” is any material which prevents orlimits energy transfer or quenching. Such materials include but are notlimited to conductive materials, high index materials, and lightimpermeable materials. In a preferred embodiment the material shield isa conductive material shield. As used herein a “conductive materialshield” is a material which is at least conductive enough to preventenergy transfer between donor and acceptor sources.

Each of the above methods of the invention are useful for at leastvarious aspects of sequencing polymers. Also all of the methods can beused with the various labeling schemes described with respect to themethod of analyzing polymers.

The methods of the invention can be accomplished using any device whichproduces a specific detectable signal for an individual unit of apolymer. One type of device which enables this type of analysis is onewhich promotes linear transfer of a polymer past an interaction stationor a signal generation station. According to one aspect of theinvention, an article of manufacture which is useful for performing themethods of the invention is provided. The article of manufactureincludes a wall material having a surface defining a channel, an agentselected from the group consisting of an electromagnetic radiationsource, a quenching source, a luminescent film layer, and a fluorescenceexcitation source, attached to the wall material adjacent to thechannel, wherein the agent is close enough to the channel and is presentin an amount sufficient to detectably interact with a partner compoundselected from the group consisting of a light emissive compound and aquencher passing through the channel.

A “wall material” as used herein is a solid or semi-solid barrier of anydimensions which is capable of supporting at least one channel. Asemi-solid material is a self supporting material and may be forinstance a gel material such as a polyacrylamide gel. For instance thewall material may be composed of a single support material which may beconducting or non-conducting, light permeable or light impermeable,clear or unclear. In some instances the agent is embedded within thewall material. In these instances the wall material can be solely orpartially made of a non-conducting layer, a light permeable layer or aclear layer to allow the agent to be exposed to the channel formed inthe wall material to allow signal generation. When the wall material isonly partially made from these materials the remaining wall material maybe made from a conducting, light impermeable or unclear layer, whichprevent signal generation. In some cases the wall material is made up oflayers of different materials. For instance, the wall material may bemade of a single conducting layer and a single non-conducting layer.Alternatively the wall material may be made of a single non-conductinglayer surrounded by two conducing layers. Multiple layers and variouscombinations of materials are encompassed by the wall material of theinvention.

As used herein a “luminescent film layer” is a film which is naturallyluminescent or made luminescent by some means of excitation orillumination, e.g., electrooptic thin films and high index filmsilluminated by internal reflection.

A “conductive material” as used herein is a material which is at leastconductive enough to prevent energy transfer between a donor and anacceptor.

A “nonconductive material” as used herein is a material which conductsless than that amount that would allow energy transfer between a donorand an acceptor.

A “light permeable material” as used herein is a material which ispermeable to light of a wavelength produced by the specificelectromagnetic radiation, quenching source, or the fluorescenceexcitation source being used.

A “light impermeable material” as used herein is a material which isimpermeable to light of a wavelength produced by the specificelectromagnetic radiation, quenching source, or the fluorescenceexcitation source being used.

A “channel” as used herein is a passageway through a medium throughwhich a polymer can pass. The channel can have any dimensions as long asa polymer is capable of passing through it. For instance the channel maybe an unbranched straight cylindrical channel or it may be a branchednetwork of interconnected winding channels. Preferably the channel is astraight nanochannel or a microchannel. A “nanochannel” as used hereinis a channel having dimensions on the order of nanometers. The averagediameter of a nanochannel is between 1 nm and 999 nm. A “microchannel”as used herein is a channel having dimensions on the order ofmicrometers. The average diameter of a microchannel is between 1 μm and1 mm. Preferred specifications and dimensions of channels usefulaccording to the invention are set forth in detail below. In a preferredembodiment, the channel is fixed in the wall.

An agent is attached to the wall material in such a manner that it willdetectably interact with a partner compound by undergoing energytransfer or quenching with the partner light emissive compound which ispassing through the channel of the wall material. In order to interactwith the partner compound the agent can be positioned in close proximityto the channel. For example, the agent may be attached to the inside ofthe channel, attached to the external surface of the wall material,attached to a concentrated region of the external surface of the wallmaterial surrounding the rim of the channel, embedded within the wallmaterial, or embedded in the form of a concentric ring in the wallmaterial surrounding the channel. Optionally the agent may cover theentire surface of the wall material or may be embedded throughout theentire wall material. In order to improve signal generation when theagent is not localized, a mask may be used to cover some areas of thewall material such that only localized regions of agent are exposed. A“mask” as used herein is an object which has openings of any size orshape. More than one agent may be attached to the wall material in orderto produce different signals when the agents are exposed to the partneragent.

The agent may be attached to the surface of the wall material by anymeans of performing attachment known in the art. Examples of methods forconjugating biomaterials are presented in Hermanson, G. T., BioconjugateTechniques, Academic Press, Inc., San Diego, 1996, which is herebyincorporated by reference.

When the agent is attached to the surface of the wall material it may beattached directly to the wall material or it may be attached via alinker. A “linker” as used herein with respect to the attachment of theagent is a molecule that tethers a light emitting compound or aquenching compound to the wall material. Linkers are well known in theart. Commonly used linkers include alkanes of various lengths.

The agent is attached to the wall material in an amount sufficient todetectably interact with a partner light emissive compound. As usedherein a “partner light emissive compound” is a light emissive compoundas defined above but which specifically interacts with and undergoesenergy transfer or quenching when positioned in close proximity to theagent. The amount of partner light emissive compound and the amount ofagent required will depend on the type of agent and light emissivecompound used.

Another example of an article of manufacture of the invention is a wallmaterial having a surface defining a plurality of channels and a stationattached to a discrete region of the wall material adjacent to at leastone of the channels, wherein the station is close enough to the channeland is present in an amount sufficient to cause a signal to arise from adetectable physical change in a polymer of linked units passing throughthe channel or in the station as the polymer is exposed to the station.A “discrete region” of the wall material adjacent to at least one of thechannels is a local area which is surrounded by wall material not havinga station. The area surrounding the discrete region does not interactwith the unit to produce the same characteristic signal produced by theinteraction of the unit with the station. The discrete region ispositioned in or near the channel such that the station at the dictreetregion is exposed to the unit as it traverses the channel.

An additional article of manufacture of the invention is a wall materialhaving a surface defining a channel and a plurality of stations eachattached to a discrete region of the wall material adjacent to thechannel, wherein the stations are close enough to the channel and arepresent in an amount sufficient to cause a signal to arise from adetectable physical change in a polymer of linked units passing throughthe channel or in the station as the polymer is exposed to the station.

As used herein a “plurality of stations” is at least two stations.Preferably a plurality of stations is at least three stations. Inanother preferred embodiment a plurality of stations is at least fivestations.

In a preferred embodiment the article of manufacture is a nanochannelplate. The following description of an optimal design of a nanochannelplate having fluorophores embedded within the plate is provided forillustrative purposes only. The example describes methods for optimizingseveral aspects of the article of manufacture. The description is in noway limiting of the article of manufacture claimed herein.

Several examples of nanochannel plates are presented in FIG. 4. FIG. 4Ashows a nanochannel plate (60) having layers of conducting material (62)and non-conducting material (64). The channel (70) has a diameter thatis sufficient to encompass the passage of double-stranded, labeled DNAin a linear fashion. In this example donor fluorophores (68) areembedded in the clear non-conducting material in a concentric ringaround each channel. The remaining portion of the nanochannel plate ismade up of a light impermeable material (66).

FIGS. 4B, C, D, E, and K show a nanochannel plate having fluorophores(68) attached to the surface of the wall material surrounding theopening produced by the channel (70). As shown in FIGS. 4B and 4D thefluorophores may cover the entire surface of the wall material. Thefluorophores may also be concentrated around the channel opening asshown in FIGS. 4C, E, and K rather than covering the entire surface.Additionally, the wall material supporting the fluorophores may be aconducting layer (62) such as that shown in FIGS. 4D and E, the wallmaterial may be a light impermeable layer (78) as shown in FIG. 4K orthe wall material may be a support layer (72) as shown in FIGS. 4B andC. A support layer may be any type of wall material including but notlimited to conducting, non-conducting, clear, light permeable, and lightimpermeable.

FIGS. 4F, G, H, I, and L show a nanochannel plate having fluorophores(68) embedded in the wall material surrounding the channel (70). Againthe fluorophores may extend across the entire wall material (shown inFIG. 4F) or may be concentrated around the channel as shown in FIGS. 4G,H, and I. In the embodiments shown in FIGS. 4F, G, H, I, and L thefluorophores are embedded in a layer of non-conducting material (62) orof a clear material (74) or of a light permeable material (79). Thelayer having the fluorophores embedded within it may form the surface ofthe wall material as shown in FIGS. 4F and G or may be sandwichedbetween other layers. For instance the non-conducting layer (64) inFIGS. 4H and I is sandwiched between two conducting layers (62). Thelight permeable layer (79) of FIG. 4L is sandwiched between two lightimpermeable layers (78). In some cases the layers shown form the entirewall material. In other cases the layers maybe adjacent to or sandwichedbetween supporting layers as shown in FIG. 41.

FIG. 4J shows a nanochannel plate having fluorophores (68) attached tothe surface of the wall material surrounding the opening formed by thechannel (70). The material surrounding all of the exposed surfaces ofthe wall material, including the surface within the channel is aconducting material.

FIGS. 4M and N show a nanochannel plate having a luminescent thin film(76) positioned within the wall material surrounding at least a portionof the channel (70). The luminescent thin film either forms the surfaceof the wall material and is adjacent to a light impermeable layer (78)as shown in FIG. 4M or may be sandwiched between two light impermeablelayers (78) as shown in FIG. 4N.

FIGS. 4O and P show a nanochannel plate having two layers offluorophores (68) either embedded in the wall material or attached tothe surface of the wall material. In FIG. 4O the fluorophore layers (68)are attached to the surface of a conducting material (62) on either sideof the wall material and surrounding the openings formed by the channel.In FIG. 4P the fluorophore layers (68) are embedded in two lightpermeable layers (79) which sandwich a light impermeable layer (78).

A preferred method of the invention involves the analysis ofradiolabeled polymers as discussed above. Preparation of radiolabeledpolymers such as DNA (for example, with ³²P or ³H) is known in the art.The following description represents one of the many possibleembodiments of analyzing radiolabeled polymers according to the methodsof the invention (FIGS. 4Q and R). A radiolabeled nucleic acid molecule(160) is analyzed with a single fabricated multilayered nanochannel(162). The nanochannel is the diameter of the labeled nucleic acidmolecule and is constructed to yield a defined region of detection. Theexemplary nanochannel plates shown in FIGS. 4Q and R are a heterogeneousmultilayered structure consisting of two radiation impermeable layerssuch as lead or Lucite films (164, 166) and a film of lower densitymaterial (or scintillation layer) (168) (i.e., conventional polymers,polymethylmethacrylate, polystryrene, Teflon, etc.). The lead films inFIG. 4Q sandwich the layer of lower density material and are of suchthickness as to prevent passage of radiation. The lower density materialpermits passage of radiation, thereby creating a defined region ofradiation detection. As the radiolabel on the DNA passes through thedefined region of detection, nuclear radiation is emitted, some of whichwill pass through the defined region of radiation detection. FIG. 4Rshows a nanochannel plate having low density material (168) surroundingthe opening formed by the channel. The material surrounding all of theexposed surfaces of the wall material including the surface within thechannel is a radiation impermeable layer (165).

In a related embodiment of analysis of radiolabeled nucleotides (FIG.5), a detection system based on scintillation counting and multiplenanochannels is presented. A nanochannel array (170) is fabricated asshown in FIG. 5. Multiple multilayered channels (172) exist for parallelamplification of data output. Each individual channel consists of twonuclear radiation shielding layers (174) which shield nuclear radiation,and a scintillation layer (176) which is fluorescently excited withexposure to nuclear radiation. The individual channels are separatedfrom each other by a nuclear radiation shielding material. The nuclearradiation is prevented from reaching the fluorescent detection system byoverlaying with optical quality Lucite (or any other transparentmaterial which prevents the passage of nuclear radiation). This allowsonly the fluorescent signal to reach the detection system.

Each of the above described nanochannels is only an example. It is,therefore, anticipated that each of the limitations described withrespect to these embodiments involving any one element or combinationsof elements can be included in each nanochannel. Preparation of filmshaving multiple layers of differing material have been described in theart, e.g., U.S. Pat. No. 5,462,467, Ferreira et. al., Thin Solid Films244:806-809 (1994).

In the example provided donor fluorophores are concentrated in aconcentric ring around each channel in order to optimize the donorintensity. Intuitively, a concentric ring is preferred because the rangeof energy transfer is limited in part by a radial Förster distance.Examining one channel in detail will illustrate the marked changes indonor intensities that can occur for the concentric ring configuration.FIG. 6 shows a single channel (82) having the concentric ringconfiguration. The outermost concentric ring (86) marks the edge of thedonor fluorophores situated around the channel. The middle concentricarea (84) denotes the region where the rate of energy transfer from thedonor to the acceptor is greater than the rate of emission of the donorfluorophores.

The rationale for embedding the donor fluorophores in a solid medium isbest understood by examining the mechanisms of photobleaching andquenching. The factors that may diminish a fluorescent signal include:photobleaching, temperature quenching, energy transfer quenching,collisional quenching, excited state reactions, and solvent effects.These mechanisms are similar in that they all arise from eithercollisional or bimolecular events. A solid medium is a physical barrierto these undesired molecular processes and is a means of isolation ofthe donor fluorophores. For example, the mechanism of photobleaching isdue to a reaction between an excited fluorophore and oxygen to form anon-fluorescent product (Menter et al., 1978; Menter et al. 1979; Gilohand Sedat, 1982). A solid medium, especially one manufactured in anoxygen-free environment, prevents oxygen from reaching the embeddedfluorophores, eliminating possible oxidative reactions (Garland andMoore, 1979; Rost, 1990). In order to understand the effect of a solidmedium further, a brief summary of the other quenching effects ispresented. Temperature quenching is a decrease in fluorescence astemperature increases. The degree of temperature dependence depends onthe compound; it is usually about 1% change in quenching per degreeCelsius (Guilbault, 1973). The effect is believed to be due to increasedmolecular motion and increased frequency of collisions, resulting inincreased probability of transition to the ground state beforefluorescence can occur. Collisional quenching is a broad categoryconsisting of many possible mechanisms involving direct contact betweena fluorophore and another molecule (Lakowicz, 1983). Excited statereactions involve the reactive excited state of a fluorophore in areaction with nearby solvent molecules and constitutes a subset ofcollisional quenching (Porter, 1967; Zweig, 1973). Solvent effectsconsists of solvent-fluorophore collisions and interactions, includingeffects of hydrogen bonding, acid-base chemistry, and charge transferinteractions (Lakowicz, 1983). Energy transfer quenching is due to theeffects of impurities on fluorophores via undesired FRET. From thisshort listing of photobleaching and quenching mechanisms, it is clearthat molecular interactions with fluorophores are the main causes ofdiminished fluorophore emissions. A solid media thus eliminates theseundesired molecular interactions, collisions, and reactions by isolatingthe fluorophores and creating a physical barrier that prevents the entryof undesired quenching molecules.

The equation defining energy transfer should be examined in order todetermine the effect of a solid medium on energy transfer. The equationthat needs to be considered describes the Förster distance (Selvin,1995; Cantor and Schimmel, 1980; Wu and Brand, 1994; Clegg et al., 1995;Fairclough and Cantor, 1978):

$\mspace{20mu} {R_{o} = \frac{\sqrt[\text{?}]{8.79 \times 10^{- \text{?}}}}{n^{4}}}$?indicates text missing or illegible when filed

J is the normalized spectral overlap of the donor emission and acceptorabsorption, φ_(D) is the quantum efficiency (or quantum yield) for donoremission in the absence of acceptor (φ_(D) is the number of photonsemitted divided by number of photons absorbed), n is the index ofrefraction, and k² is the geometric factor related to the relative angleof the two transition dipoles. Embedding the donor fluorophore in asolid medium raises n, the index of refraction. In solvent systems, n istaken to be around 1.35 (Selvin, 1995), slightly higher than the valuefor water (1.33). Some typical values for solid media are 1.46 for fusedquartz, 1.52 for crown glass, and 1.66 for dense flint glass. A ratio ofthe R_(o)'s for a solid medium and a solvent system can be used todetermine the magnitude of the effect of changing the index ofrefraction:

$\frac{R_{o}}{R_{o}} = \left( \frac{n}{n} \right)^{23}$

where the primed values are the ones for the solid medium and theunprimed ones are for a solvent system. Assuming n′=1.5, the ratiobecomes 0.93. The Förster distance thus only changes by 7%, a value thatcan be easily corrected for by using donor-acceptor pairs that have ahigher original R_(o).

Referring again to FIG. 6, conceptually, without presenting equations,the rate of energy transfer (k_(transfer)) from the center of thechannel falls off with increasing distance from the center (FIG. 6). Itis also known that the rate of donor emission (k_(emit)) is uniform overthe complete area of donor fluorophores in the absence of acceptors(FIG. 6). The area closest to the channel, and thus to the acceptor, isquenched completely. At all areas less than R_(equal), the donorfluorophore is quenched completely because k_(transfer) is greater thank_(emit). At areas greater than the R_(equal), the rate of donoremission is greater than the rate of energy transfer so quenching isincomplete.

If the concentric ring of donor fluorophores has a radius equal toR_(equal), then a hundred percent intensity change can occur for thedonor fluorophores. This means that upon the exit of an acceptor labelednucleotide through the channel, the acceptor is detected with perfectefficiency. Recalling that confidence intervals are related to the SNRof the system, the minimum signal to noise ratio needed to generate a99.9% confidence interval for such a change is calculated to be 6:1. TheSNRs for the detection systems proposed are greater than 600:1.

A quantitative explanation is presented at this time to calculateR_(equal). The equations for the rates of donor emission and energytransfer are presented. The rate of donor emission (k_(emit)) is asfollows:

$k_{emit} = \frac{I \in {\rho \; N}}{hv}$

where I is the intensity of light incident upon the donor fluorophores,e is the molar extinction coefficient of the donor fluorophore, r isconstant for fluorescence emission (3.8×10⁻²¹ mol cm³/L), N is thenumber of donor fluorophores, h is Planck's constant (6.6261×10⁻³⁴ J s),and u is the frequency of excitation light.

The rate of energy transfer (k_(transfer)) from many donors to oneacceptors is derived from the original Förster rate equation (Förster,1965) for one donor and one acceptor. The original equation is given as:

$K = {\frac{1}{\tau_{D}}{\langle\frac{R_{o}}{r}\rangle}^{6}}$

where k is the rate of energy transfer from one donor to one acceptor,R_(o) is the Förster distance, τ is the fluorescence lifetime of thedonor, and r is the distance from the donor to the acceptor. Thederivation of a multi-donor system is straightforward and follows fromthe discussion of the multi-donor system described above.

There are two limitations in the amount of energy transferred for amulti-donor system. First, there is the saturation limit imposed by thelifetime of the acceptor. The acceptor is only able to be excitedwhenever it is in the ground singlet state. For an acceptor with alifetime of 1 ns, the upper limit is 1×10⁹ excitation events/second.This large saturation level is hardly a concern given the low rate ofexcitation for a single fluorophore (25,000 excitation events/second).The second limitation is the probability of simultaneous de-excitationof donor fluorophores. As calculated above only a very small number ofsimultaneous de-excitation events can occur. Since the acceptor is notsaturated with excitation events resulting from energy transfer and theprobability of simultaneous donor de-excitation is small, the rate ofenergy transfer for a multi-donor system is directly proportional to thenumber of donor fluorophores (N):

$K_{transfer} = {\frac{N}{\tau_{D}}{\langle\frac{R_{o}}{r}\rangle}^{6}}$

With this equation, the radius at which K_(transfer) is equal tok_(emit) (R_(equal)) is found by equating the two rate equations andsolving for r.

$K_{emit} = {\left. K_{transfer}\Rightarrow\frac{I \in {\rho \; N}}{hv} \right. = {\frac{N}{\tau_{D}}\left( \frac{R_{o}}{r} \right)^{6}}}$

-   -   Solve for r.

$\mspace{20mu} {R = {R_{equal} = \frac{R_{o}}{\sqrt[\text{?}]{\text{?}}}}}$?indicates text missing or illegible when filed

A numerical value for R_(equal) can be calculated. Table 3 lists thevalues for the variables and the reason the particular value is chosen.

TABLE 3 variable value reason R_(o)  60 Å range for Förster is 20 Å-100Å τ_(D) 1 × 10⁻⁹ s/photon fluorescent lifetimes range from 1 ns-20 ns h6.6261 × 10⁻³⁴ J s Planck's constant v 6.1224 × 10¹⁴ × s⁻¹ C = vλ; λ =490 nm (excitation of fluorescein) I 30 W/cm² intensity of 2 W laser isgiven by P/A. A = beam area (r = 2 mm). Intensity of laser is 64 W/cm² ε91,0001/M cm molar extinction coefficient for fluorescein ρ 3.8 × 10−21M cm³ constant for fluorescence emission of fluorescein R_(equal) 350 Åfrom above equation

R_(equal) is calculated to be 350 Å, within an order of magnitude of theFörster distance. This means that a concentric ring of fluorophorearound a channel with a radius equal to 350 Å will give rise to ahundred percent change in signal intensity upon the passage of anacceptor label. In practice, having such a large donor intensitydecrease is unnecessary from both the standpoint of signal detection andthe need to resolve adjacent bases. For example, with a SNR of 600:1,only a 0.50% intensity change will give rise to a 99.9% confidenceinterval. Resolution between adjacent bases is possible by looking atfurther decreases in the donor emission when there are two acceptorsinstead of one in the donor layer. If one acceptor already decreases thedonor emission to zero, then an additional acceptor will not be detectedbecause the donor emission cannot decrease further. This aspect isdiscussed in detail below. An example of a method for constructing aconcentric ring of donor fluorophores around each channel by using aphotolabile protecting group and light diffraction is also providedbelow.

Nanochannels having a channel diameter size of at least 1 nm arecommercially obtainable in the form of polycarbonate filters fromPoretics, Inc. and can be made on order by Corning Separations, Inc.There are several methods that can be used to create nanochannels of thedesired diameter.

One method for preparing a nanochannel plate is by a track-etchprocedure which produces cylindrical pores of uniform diameter in amembrane material. Microporous and nanoporous polymeric membranes havingpore diameters on the order of 10 nm and with pore densities approaching10⁹ pores per square centimeter can be prepared by the track-etch method(R. L. Fleischer, P. B. Price, R. M. Walker, Nuclear Tracks in Solids(Univ. of California Press, Berkeley, Calif. (1975)). The manufacture ofpores via track-etch is a two step process. In the first step, thinpolycarbonate (or other polymeric material) film is exposed tocollimated, charged particles in a nuclear reactor. As these particlespass through the polycarbonate material, they leave sensitized tracks.The density of the tracks is controlled by varying the amount of timethe film is in the reactor. In the second step, the tracks left by theparticles are preferentially etched, or dissolved, into uniform,cylindrical channels. The diameters of the perforations can becontrolled by the residence time of the etchant on the film. Manyexamples of methods for forming track etched membranes have beendescribed in the art, e.g. European patent Application No. 83305268.1,Publication No. 0109147, to Varian Associates, Inc., U.S. Pat. Nos.3,303,085; 3,662,178; 3,713,921; 3,802,972; 3,852,134, 4,956,219,5,462,467, 5,564,959 and 5,449,917, each of which is incorporated hereinby reference.

The commercially available membranes are generally prepared frompolycarbonates or polyesters; however, a number of other materials areamenable to the track-etch process (Id.). For instance, other polymericmaterials include but are not limited to polystyrenes, aromaticpolyesters, polyolefins, including polyethylene, polyethyleneterephthalate, polypropylene, vinyl plastics such as polyvinyldifluoride (PVDF), and cellulose esters such as cellulose nitrate,cellulose butyrate and cellulose acetate. If the nanochannel plate ofthe invention is prepared by a track-etch technique it may be formedfrom any material capable of being track etched can be used to form thetrack etched membrane.

Devices for performing bombardment of materials with high energyparticles are well known in research and industry. The particles used toform the tracks may be generated by a charged particle accelerator, suchas an electrostatic accelerator (e.g., a Van de Graaff accelerator orTandem accelerator), a linear accelerator or a cyclic accelerator suchas a cyclotron or any other means known in the art.

Once the damaged track is formed in the film the channels or pores areformed by selectively etching the film with a gas or liquid. Theresidence time of the etchant determines the size of the channels. Thetrack etched film is exposed to the etchant for sufficient time togenerate channels that are sized to match the desired application, whichvaries depending on the type of polymer being analyzed and the type ofanalysis. The channel diameter can be measured using a scanning electronmicroscopy (SEM) according to methods disclosed in Basic Principles ofMembrane Technology, M. Mulder, Klumer Academic, 1991.

A second method of creating nanochannels of defined diameter is to use acombination of track-etching and surface coating. A polycarbonatemembrane of a diameter greater than the desired nanochannel device iscoated with a thin film of material with a defined thickness. Theresulting structure is a polycarbonate membrane surface coated to thedesired diameter. The first layer of thin film that is added to thenanochannel plate is a conducting layer. A conducting layer helps toresolve adjacent bases (which is discussed below in more detail).

Thin layers of conducting polymers are added to the polycarbonatemembrane through solvent deposition. Solvent deposition of conductingpolymers have been described (Cheung et al., 1994; Fereira et al., 1994;Fereira and Rubner, 1995; Fou and Rubner, 1995). The following isexcerpted from Fou and Rubner, 1995:

-   -   We describe the solution chemistry and methodologies needed to        utilize the layer-by-layer processing technique described . . .        to manipulate conducting polymers such as polypyrrole and        polyaniline into multilayer thin films with angstrom-level        control over both film thickness and film architecture.        Ultrathin films with conductivities over 300 S/cm can be made.        The process involves the spontaneous adsorption of monolayers of        electrically conductive polymers onto substrates from dilute        solutions. Subsequent multilayer thin films are created by        alternate deposition with soluble polyanions. The thickness of        the thin films can be precisely controlled to the angstrom level        and can range between 5 Å and greater than 1000 Å. The        conductive polymers used are polypyrrole and polyaniline because        these can be made extremely conductive (300 S/cm) for ultrathin        layers (˜50 Å).

The advantage of using a solvent deposition method to create the desirednanochannels is two-fold. First, nanochannels of any particular diametercan be created with precision and accuracy. Second, since the firstlayer of the nanochannel plate is a conducting layer, it is onlyconvenient to add it. An additional advantage, though not significant,is that addition of a thick layer of conducting material can lower thepeak-to-valley distance of the polycarbonate surface, creating a moreuniform surface.

Another method for preparing a nanochannel device of the invention isthrough the production of arrays of carbon nanotubes. Iijimademonstrated the production of multiple concentric cylindrical shells ofhexagonally bonded carbon atoms which can serve as catalytic surfaces toconfine species in a 1-dimensional space. Iijima, Nature, 354:56 (1991),see also U.S. Pat. No. 4,424,054.

Li, W. Z., et. al., has also reported a method for producing large areasof highly ordered, isolated long carbon nanotubes. The method is basedon a chemical vapor deposition which utilizes mesoporous silicacontaining iron nanoparticles embedded in the pores rather than carbonblack and graphite or silica covered with transition metalnanoparticles. The following method is disclosed in Li, W. Z. et al.:

-   -   Mesoporous silica containing iron nanoparticles were prepared by        sol-gel process from tetraethoxysilane (TEOS) hydrolysis in iron        nitrate aqueous solution.

Analytically pure TEOS (10 ml) was mixed with 10.4 ml of analyticallypure ethyl alcohol and 0.1 M iron nitrate aqueous solution (11.4 ml) bymagnetic stirring for ˜30 min. A few drops of concentrated hydrogenfluoride (0.2 ml) were then added, and the mixture was stirred for 15min. After gelation of the mixture, the gel was dried for 1 week at 60°C. to remove the excess water and other solvents. The gel was thencalcined 10 hours at 450° C. at 10⁻² torr. A silica network withrelatively uniform pores was obtained with iron oxide nanoparticlesembedded in the pores. The iron oxide nanoparticles were then reduced at550° C. in 180 ton of flowing 9% H₂/N₂ (110 cm³/min) for 5 hours toobtain iron nanoparticles, which have a high catalytic activity.Subsequently, a mixture of 9% acetylene in nitrogen was introduced intothe chamber at a flow rate of 110 cm³/min, and carbon nanotubes wereformed on the substrate by deposition of carbon atoms obtained fromdecomposition of acetylene at 700° C. The samples were examined by ascanning electron microscope (SEM) (S-4200, Hitachi) before and aftercarbon deposition, and energy-dispersive x-ray spectra (EDX) wererecorded by a SiLi detector attached to the SEM. To prepare atransmission electron microscope (TEM) specimen, the sample was groundin a mortar and suspended in ethanol; a drop was then placed on a holeycarbon copper grid and examined in a JEM 200-cx microscope tocharacterize the carbon nanotubes.

Additionally, nanochannels may be prepared from anodic porous aluminawhich is a packed array of columnar hexagonal cells with central,cylindrical, uniformly sized holes typically ranging from 4 to 200 nm indiameter. Membranes of this type are prepared electrochemically fromAluminum metal (A. Despic and V. P. Parkhutik, in Modern Aspects ofElectrochemistry, J. O. Bockris, R. E. White, B. E. Conway, Eds.(Plenum, New York, 1989), vol. 20, chap. 6.). Pore densities as high as1011 pores per square centimeter have been achieved (D. AlMawiawi, N.Coombs, M. Moskovits, J. Appl. Phys. 70, 4421 (1991)). Membranes havingpore diameters as small as 5 nm have been prepared using this method(and it is believed that even smaller pores can be prepared). Martin, C.R., Science, 266:1961 (1994).

Matsuda and Fukuda have described a modification of porous aluminamembranes. The membranes which are a highly ordered metal (platinum andgold) nanohole array are prepared using a two-step replication of thehoneycomb structure of anodic porous alumina. Preparation of thenegative porous structure of porous alumina followed by the formation ofthe positive structure with metal results in a geometrical structureidentical to that of anodic porous alumina. The method, therefore allowsthe preparation of the hole array of anodic porous alumina with desiredmaterials other than alumina. Matsuda and Fukuda, Science, 268:1466(1995). Matsuda and Fukuda's procedure is summarized below.

Anodic alumina was produced using a long period anodization of aluminaon a substrate at 40 V which results in a minimum number of defects anddislocations, followed by removal of the aluminum substrate and thebottom part of the porous layer with saturated HgCl₂. The material wasthen dipped in 5% (by weight) phosphoric acid solution at 30° C. toadjust the pore diameter. A thin metal layer was deposited on the bottomof anodic porous alumina by vacuum deposition in order to create acatalyst or electrode for the subsequent metal-plating process.Generally the same metal as that used to create the scaffold is used forthe evaporation. Metal methacrylate monomer containing a polymerizationinitiator such as 5% (by weight) benzoyl peroxide was injected into theholes under vacuum conditions and was polymerized by ultravioletirradiation. The alumina layer was then removed with NaOH to produce anegative porous alumina-type structure of poly(methyl methacrylate)(PMMA). A positive structure was formed from the PMMA mold byelectroless deposition of platinum as follows. The negative type of PMMAwas dipped into the electroless plating solution, causing metaldeposition to start at the bottom part of the cylindrical structure andthe metal to gradually fill the cavity of the PMMA to the top of thenegative type of PMMA. Alternatively a gold (Au) hole array was preparedusing electrochemical deposition of Au into the microcavity of the PMMAunder constant conditions. The porous metal was obtained by dissolvingthe PMMA with acetone.

Other nanoporous materials which have been described in the art includea nanochannel array glass with pore densities as high as 3×10¹⁰ poresper square centimeter (R. J. Tonucci, B. L. Justus, A. J. Campillo, C.E. Ford, Science 258, 783 (1992) and Pearson and Tonucci, Science, 270:68 (1995)). Douglas et al. have shown that the nanoscopic pores in aprotein derived from a bacterium can be used to transfer an image ofthese pores to an underlying substrate (Douglas, et. Al., Science 257:642 (1992)). Finally, Ozin has discussed a wide variety of nanoporoussolids that can be used as template materials (Ozin, G. Adv. Mater.4:612 (1992)). Nishizawa et. al., describe the production of metalnanotubule membranes having radii as small as 0.8 nm (Nishizawa et. al.,Science 268:700 (1995), describing nanotubules formed by plating goldonto the walls of pores in a commercially available polycarbonatefiltration membrane (Poretics) containing cylindrical nanopores ofuniform radius (25 nm, 6×10⁸ pores per square centimeter) runningthrough the complete thickness (6 μm) of the membrane. “The thickness ofthe Au layers deposited on the pore walls can be controlled by varyingthe plating time. As a result, the inside radius of the Au nanotubulescan be varied at will [as determined from measurements of gas (He) fluxacross the tubule-containing membrane.]” Berggren et. al., havedemonstrated techniques for nanolithography using self-assembledmonolayers and a beam of neutral inert gas. Berggren et. al., Science269: 1255-1257 (1995).

La Silva et al., describe a technique for fabricating simple metalstructures with a scanning tunneling microscope (STM) which havedimensions of 10's to 100's of nanometers and are partially electricallyisolated from their environment. The method is performed by depositing avery thin metal film on an insulating substrate, and using the tip tomachine gaps through the film where lateral electrical insulation isdesired. (Journal of Vacuum Science & Technology B, (1993)11:1992-1999).

The wall material may be constructed in a manner which is optimal forresolving adjacent units of the polymer. Since the purpose of the wallmaterial is to provide an environment which is conducive to generating asignal, the materials used to prepare the wall material may be selectedto aid in this process. For instance, the wall material surrounding theagent if the agent is embedded in the wall material preferably is anon-conducting or light permeable material. At least two othermechanisms for optimizing the wall material in an apparatus havingfluorophores embedded in the wall surrounding the channel, in order toresolve adjacent labeled bases include the use of thin conducting layersand controlling the radii of donor fluorophores around each channel.

Conducting layers prevent Förster energy transfer through electricalshielding. FIG. 6 demonstrates a configuration of the conducting layersrelative to the non-conducting layer which contain the donorfluorophores. The conducting “sandwich” creates a defined region whereenergy transfer can occur and this helps to optimize base resolution.Förster energy transfer arises because of an electrical dipole-dipoleinteraction between a donor and acceptor. The rationale for the use ofconducting layers when the signal generated is dependnet on FRET is thatFRET is electric field-dependent and thus placing an electric shieldbetween the donor and acceptor will prevent energy transfer. It isnecessary to understand fully the basis of energy transfer. Incidentexcitation light creates an electric field in the donor because thelight induces transitions in the donor, or causes electrons in the donorto oscillate (Selvin, 1995). This creates an induced, electric dipolemoment in the donor, which in turn, creates its own electric field.Energy transfer arises when an acceptor is placed in the donor'selectric field. As a consequence, there are induced transitions in theacceptor which create an induced dipole moment, ρ_(A). The size of thedipole moment is related to the size of the donor electric field:ρ_(A)=α_(A)E_(D), where α_(A) is the polarizability of the acceptor. Theamount of energy absorbed by the acceptor is ρ_(A)E_(D), =α_(A)E_(D) ²which translates into the 1/R⁶ dependence of the rate of energytransfer.

The theory of electrical shielding is found in most introductory physicsbooks. Purcell, 1985 provides a clear explanation. The potentialfunction inside the box, Ψ. (x, y, z), must satisfy Laplace's equation,▾²Ψ=0. Given the knowledge of conductors, the boundary of the conductoris an equipotential, meaning that Ψ=Ψ_(o) a constant function everywhereon the surface of the conductor. An obvious solution to Laplace'sequation is Ψ=Ψ_(o) throughout the volume. According to the uniquenesstheorem, there can only be one solution, meaning the answer is Ψ=aconstant. The electric field of a constant potential function is zerobecause E=−grad Ψ. The electric field is thus zero everywhere inside thebox.

The model of a box with an open hole can be applied to a nanochannelplate because essentially a nanochannel plate is a conducting box withmany “holes”. Consider the FIGS. 7A and 7B. FIG. 7A shows a completenanochannel plate having nanchannels (90). The layer of donorfluorophores (88) is enclosed by the conducting “box.” In this manner,there cannot be energy transfer to the donor fluorophores from outsidethe box because of electrical shielding (92). Recall that the basis ofenergy transfer is electric. At the boundary condition of thenanochannel, which is the outer surface of the nanochannel plate,Ψ=Ψ_(o), an equipotential surface. A logical solution to Laplace'sequation is that everywhere inside the boundary condition that,Ψ=constant. From the uniqueness theorem, this is the only possibleanswer and thus everywhere inside the conducting nanochannel “box,” E=0because E=Ψ-grad. The conclusion is that acceptor fluorophores cannotundergo energy transfer from outside the box. Even inside a nanochannel,as shown in FIG. 7B, energy transfer is limited geometrically. At anyposition where the acceptor molecule is not in the plane of the donorfluorophores, the amount of energy transfer is limited. In FIG. 7B, theamount of donor fluorophores that cannot undergo energy transfer isenclosed by an angle of θ, which for all purposes is very large unlessthe acceptor fluorophore is directly in the plane of the donorfluorophores, which is the desired configuration.

The consequence of the conductive layers is that a sharp signal iscreated upon the passage of a labeled nucleotide through thenanochannel. FIG. 8A and FIG. 8B demonstrate signal generation uponpassage of an acceptor label through the nanochannel. FIG. 8A shows anenlarged view of one nanochannel (98). Only part of the conductinglayers (94) is shown. The light impermeable polycarbonate layer is notshown. An acceptor label (100) on a strand of DNA moves through thenanochannel from bottom to top, starting at position A and moving toposition C. FRET can only occur at position B because the conductinglayers shield any interaction of the donor fluorophores (96) with theacceptor at positions A and C. FIG. 8B graphically illustrates the sharpchanges in donor intensity as an acceptor label moves from positions Ato C. The middle region on the graph denotes the level of the donorfluorophores. The outer regions on the graph denote the level of theconducting layer. At the interface of the conducting and donorfluorophore layers, there are dramatic changes in donor intensity due toelectrical shielding.

It is clear that resolution between adjacent bases can be resolved withconducting layers. By creating a conducting “sandwich” where thethickness the donor fluorophores is less than the helical rise of B-DNA(3.4 Å), the desired resolution can be achieved. Thin films of thisthickness can be constructed easily with plasma, solution, chemicalvapor, or ion beam deposition methods (Spohr, 1990; Valiev, 1992;Konuma, 1992; Pauleau, 1995; Bruno et al., 1995; Dash, 1975; Stuart,1983; Morosanu, 1990) However, it is not desirable to use a donorfluorophore film less than 3.4 Å, as will be explained under the nextheading. In brief, donor fluorophores embedded in a thicker layer allowsthe measurement of instantaneous rate of movement of DNA. By measuringthe time a labeled nucleotide spends in the thick layer and knowing thedimensions of the layer, the rate of DNA movement is known, which isimportant for determining distances between labeled nucleotides.

Another example of a method for precisely resolving adjacent labeledbases is to control the radii of the donor fluorophores around eachnanochannel. The amount of energy transferred for two acceptor labels inthe presence of a concentric ring of donor fluorophores is greater thanthe energy transferred for one acceptor label. Detection of thedifference in energy transferred for one and two acceptors allows theresolution of adjacent bases. In order for the donor fluorophores to beable to interact with more than one acceptor, the thickness of the donorfluorophores has to be greater than the helical pitch of DNA.Furthermore, the radii of the donor fluorophores must be greater thanR_(equal) for one acceptor (see FIG. 6). A radii greater than R_(equal)for one acceptor allows for further decreases in donor intensity in thepresence of more than one acceptor. A radii at R_(equal) means that uponthat passage of one acceptor, the donor intensity decrease is equal to100%. In this case, passage of two adjacent acceptors gives the samedetected signal as one acceptor.

FIG. 9 schematically demonstrates the passage of a two-base labeledstrand of DNA (102) through a nanochannel (104) with the properthickness and radii of donor fluorophores (108) sandwiched betweenconducting material (106). The positions labeled “A” through “D”correspond to the labels on the graph shown at the right of theillustrations. Initially, the acceptor labels on the DNA are at positionA. Energy transfer is not possible at this position so donor intensityremains at a maximum. Further movement of the DNA allows one acceptor toundergo energy transfer with the donor fluorophores (B) and a sharpdecrease in the donor intensity occurs. At position C, two fluorophorescan undergo energy transfer yielding a further decrease in the donorintensity. Finally, the two acceptor labels exit the region of donorfluorophores, energy transfer is not longer possible, and the donorintensity returns to a maximum (D).

The change in donor emission in the presence of one and two acceptorscan be visually demonstrated without mathematical quantitation. FIG. 10Aillustrates the amount of change as volumes. The illustrations show theamount of energy transfer as a solid volume (110). The original donorintensity is represented as the volume in the shape of a disc (112).FIG. 10B shows the change for one acceptor. The right illustration isfor two acceptors. The decay curves represent the rate of energytransfer with respect to radial distance as given by Forster's equation.The decay curve for two acceptors is roughly double that of oneacceptor. The rates of donor emission and energy transfer are expressedin units of 1/s nm². Integration of the rate of donor emission decreaseover the surface area of energy transfer yields the net decrease indonor emission. This is represented as the shaded area under each decaycurve integrated over 2π. Knowing the original donor emission (shadedrectangular area integrated over 2π), the percent decrease in donoremission can be found.

Mathematically, the changes can be easily calculated. In both cases, thechange is equal to the striped area (FIG. 10) integrated over 2π. Tocalculate, the rates of donor emission and energy transfer have to beexpressed in the appropriate units of 1/s nm². To do so, the density ofthe donor fluorophores, given as N/A, is to be used in the rateequations, where N_(D) is the number of fluorophores and A is the areawhich is occupied by the donor fluorophores. Accordingly, the rate ofemission is given as:

$K_{emit} = \frac{I \in {\rho \; N_{D}}}{hvA}$

The rate of energy transfer becomes:

$k_{transfer} = {\frac{N_{D}N_{A}}{\tau \; D^{A}}\left( \frac{R_{o}}{r} \right)^{6}}$

Where N_(A) is the number of acceptors that can undergo energy transferwith the donors. The general equation for the striped areas integratedover 2π follows:

Π(R ² _(equal) −R ² _(channel))K _(emit)+∫_(o) ^(2πR donor)∫_(R)_(equal) k _(transfer) rdrdθ

For the present calculations, R_(donor)=55 nm, N_(D)=1000. The donorfluorophore density (N_(D)/A) becomes 0.11 fluorophores/nm². This valueis not unreasonable because the size of the area of the largest possiblefluorophore is 1 nm². This means that the density is at least an orderof magnitude lower than the highest possible fluorophore density.

In order to solve the general equation, recall that R_(equal) can besolved by setting k_(emit)=k_(transfer), resulting in the following:

$R_{equal} = \frac{Ro}{\sqrt[\sigma]{{\tau \; {DI}} \in {\rho/{NAhv}}}}$

It is also important to know the original donor emission (E_(o)), givenas:

E _(o) =k _(emit)π(R ² _(donor) −R ² _(channel))

Using the values from table 3 and from above, it is possible to tabulatedonor emission values for different numbers of acceptors that are in theposition to undergo energy transfer (table 4). Values for N_(A)=1-5,11-12 are calculated as examples. It is expected that the value of thefirst donor emission decrease be the greatest. Subsequent decreasesbecome progessively smaller. When R_(equal) approaches R_(donor), it isexpected that actual (not percent) change approaches zero. This is sobecause essentially the donor molecules are almost completely quenched.

The percent changes and the signal-to-noise ratios determine thedetection capability, not the absolute numerical changes in donoremission. As expected, the SNR decreases as the number of acceptorsincrease because there is greater donor quenching. Otherwise stated, thedonor emission becomes smaller. This decrease in SNR is compensated byincreasing percent changes from N_(A)=5 to 12. The confidence iscalculated by using the SNR and the percent change. For example, theconfidence for detecting the change from one to two acceptors uses theSNR for one acceptor and the percent change from one to two acceptors.In this case, there is a 95% confidence for detecting a 0.483% change.Since the percent change is high, 29.1%, the signal change from one totwo acceptors is detected with a 100% confidence. The calculations fromabove demonstrate that multiple adjacent acceptors can be detected withhigh efficiency.

TABLE 4 donor emission % change from SNR (80% full well N_(A)(photons/s) previous capacity at Eo, N_(A) = 0 confidence 0 2.5572 × 10⁷— 632:1 ~100% 1 1.0943 × 10⁷ 57.2% 413:1 ″ 2 7.7510 × 10⁶ 29.1% 348:1 ″3 5.7650 × 10⁶ 25.6% 300:1 ″ 4 4.3560 × 10⁶ 24.4% 261:1 ″ 5 3.2440 × 10⁶25.5% 225:1 ″ ″ ″ ″ ″ ″ 11 3.8200 × 10⁵ —  77:1 ″ 12 2.1200 × 10⁵ 44.5% 58:1 ″

An assumption made in the above calculations is that the donor emissionremains constant during the passage of one fluorophore through the donorfluorophore layer. Recall the donor fluorophore layer may consist of amonolayer of fluorophores embedded in a non-conducting medium. The rangeof energy transfer for an acceptor close to the exit of the nanochannelis less than the range upon initial entry into the channel. This changeis in fact significant, as shown mathematically below. Taking intoaccount the change yields higher signal-to-noise ratios and greaterchanges in donor intensity for additional acceptors. This means that thevalues in table 4 which show very efficient signal generation/detectionalready are actually even slightly higher.

The change in donor emission upon passage of an acceptor through thedonor fluorophore layer is determined by calculation in the followingformulas. It is expected that the amount of energy transfer decreases asan acceptor passes through the donor layer due to a smaller effectiveenergy transfer range. The consequence of this decrease is that thedonor emission is greater than in the previous calculations. A higherdonor emission means a higher SNR. Suppose an acceptor has entered thedonor layer a small distance. A short time later, another acceptorenters the donor layer. The presence of an additional acceptor yields adecrease in the donor emission. The percentage change is large. In fact,it is larger than the previous calculations. The combination of higherSNRs and greater percentage changes mean that detection efficiencies aregreater than those previously estimated. Complex patterns of labeling,such as labeling every several nucleotides, can be distinguished usingthis system.

The equation of donor emission when the acceptor is on the same plane asthe donor fluorophores is given as:

K _(emit)π(R ² _(donor) −R _(channel) ²)−[k _(emit)π(R _(equal) ² −R_(transfre) ²)+∫_(o) ^(2πdonor)∫_(R) _(equal) k _(transfer) rdrdθ]

Recall the equations for K_(emit) and k_(transfer):

$K_{emit} = \frac{I \in {\rho \; N_{D}}}{hvA}$$k_{transfer} = {\frac{N_{D}N_{A}}{\tau \; D^{A}}\left( \frac{R_{o}}{r} \right)^{6}}$

Express the original donor emission function in terms of the radialdistance (x) and distance of acceptor from donor layer (d) with thefollowing substitutions:

${dr} = {\frac{x}{\sqrt{{x\; 2} + {d\; 2}}}{dx}}$$r = \sqrt{{x\; 2} + {d\; 2}}$

The resulting equation, together with the substitution of the equationfor k_(transfer) yields:

$\mspace{20mu} {{K_{emit}{\pi \left( {R_{donor}^{2} - R_{channel}^{2}} \right)}} - \left\lbrack {\begin{matrix}{{k_{emit}{\pi \left( {X_{equal}^{2} - R_{channel}^{2}} \right)}} +} \\{2{\pi\left( \frac{N_{D}R_{\text{?}}^{\text{?}}}{\tau \; D^{A}} \right)}{\int_{X_{equal}}^{R_{donor}}{\left( \frac{x}{x^{2} + d^{2}} \right){x}}}}\end{matrix}\text{?}\text{indicates text missing or illegible when filed}} \right.}$

Let u=x²+d². It follows that dx=du/dx.

$\mspace{20mu} {{K_{emit}{\pi \left( {R_{donor}^{2} - R_{channel}^{2}} \right)}} - \begin{bmatrix}{{k_{emit}{\pi \left( {X_{equal}^{2} - R_{channel}^{2}} \right)}} +} \\{2\pi \frac{N_{D}\pi \; R_{\text{?}}^{\text{?}}}{\tau \; D^{A}}{\int_{U - X_{equal}^{2} + d^{2}}^{U - R_{donor}^{2} + d^{2}}\left( {\frac{1}{2u^{2}}{u}} \right.}}\end{bmatrix}}$$\mspace{20mu} {{K_{emit}{\pi \left( {R_{donor}^{2} - R_{channel}^{2}} \right)}} - \begin{bmatrix}{{k_{emit}{\pi \left( {X_{equal}^{2} - R_{channel}^{2}} \right)}} +} \\{\left( {2\pi \frac{N_{D}\pi \; R_{\text{?}}^{\text{?}}}{\tau \; {DA}}} \right)\begin{pmatrix}{\frac{1}{4\left( {x_{equal}^{2} + {d\; 2}} \right)2} -} \\\frac{1}{4\left( {R_{donor}^{2} + {d\; 2}} \right)2}\end{pmatrix}}\end{bmatrix}}$ ?indicates text missing or illegible when filed

Substitute x equal=√R² _(equal)−d² and the resulting emission functionbecomes:

${E(d)} = {{K_{emit}{\pi \left( {R_{donor}^{2} - R_{channel}^{2}} \right)}} - \begin{bmatrix}{{k_{emit}{\pi \left( {R_{equal}^{2} - d^{2} - R_{channel}^{2}} \right)}} +} \\{\left( {2\pi \frac{N_{D}R_{\text{?}}^{\text{?}}}{\tau \; {DA}}} \right)\begin{pmatrix}{\frac{1}{4R_{equal}^{4}} -} \\\frac{1}{4\left( {R_{donor}^{2} + {d\; 2}} \right)^{2}}\end{pmatrix}}\end{bmatrix}}$ ?indicates text missing or illegible when filed

The resulting donor emission function can be plotted versus distance.

Donor Emission Distance (photons/s) % Change from Original 0 1.09425 ×10⁷ — 20 Å 1.09740 × 10⁷ 0.023% 40 Å  1.0692 × 10⁷ 1.158% 60 Å 1.12278 ×10⁷ 2.607% 80 Å 1.14501 × 10⁷ 4.638% 100 Å  1.17365 × 10⁷ 7.256%

${E_{2}(d)} = {{K_{emit}{\pi \left( {R_{donor}^{2} - R_{channel}^{2}} \right)}} - {\quad{\begin{bmatrix}{{k_{emit}{\pi \left( {X_{equal}^{2} - R_{channel}^{2}} \right)}} +} \\{\left( {2\pi \frac{N_{D}R_{\text{?}}^{\text{?}}}{\tau \; {DA}}} \right)\begin{pmatrix}{\frac{1}{4\left( R_{{equal} + d^{2}}^{2} \right)^{2}} -} \\\frac{1}{4\left( {R_{donor}^{2} + d^{2}} \right)}\end{pmatrix}}\end{bmatrix} + {\left( \frac{2\pi \; N_{D}R_{\text{?}}^{\text{?}}}{\tau \; {DA}} \right)\left( {\frac{1}{4R_{equal}^{4}} - \frac{1}{4R_{donor}^{4}}} \right)\text{?}\text{indicates text missing or illegible when filed}}}}}$

In order to find solutions to the above equation, R_(equal) needs to befound by equating k_(emit) to the sum of energy transfer of the twoacceptors. Solving for R_(equal) in the below equality is done bycomputer:

$k_{emit} = {\left. {k_{{transfer}{(1)}} + k_{{transfer}{(2)}}}\Rightarrow\frac{I \in {\rho \; N_{D}}}{hv} \right. = {{\frac{N_{D}}{\tau \; D}\left( \frac{R_{o}}{R_{equal}} \right)^{6}} + {\frac{N_{D}}{\tau \; D}\frac{R_{\text{?}}^{\text{?}}}{\left( {R_{equal}^{2} + d^{2}} \right)^{2}}}}}$?indicates text missing or illegible when filed

The percent change in donor emission from one to two acceptors istabulated.

Distance R_(equal) 2 % (d) acceptos E₂(d) E₁(d) change SNR  O Å 39.287.7510 × 10⁸ 1.0943 × 10⁷ 29.10% 348.0:1 20 Å 39.21 7.7661 × 10⁸ 1.0974× 10  29.24% 348.3:1 40 Å 39.11 7.8102 × 10⁸ 1.1069 × 10⁷ 29.44% 349.3:160 Å 39.01 7.8815 × 10⁸ 1.1228 × 10⁷ 29.80% 350.9:1 80 Å 38.82 7.9790 ×10⁸ 1.1450 × 10⁷ 30.31% 353.1:1 100 Å  38.610 8.0962 × 10⁸ 1.1737 × 10⁷31.01% 355.1:1

${\% \mspace{14mu} {change}} = {\frac{{E_{1}(d)} - {E_{2}(d)}}{E_{1d}} \times 100}$

FIG. 16 includes a graph illustrating the relationship of emissions todistance (Angstroms).

Therefore base resolution down to individual bases can be achieved. Acorollary to the base resolution argument is that the time spent in thedonor layer by an individual acceptor can be determined. This translatesinto information about instantaneous rates of DNA movement.

In order to achieve optimal linear crossing of a polymer across achannel it is important to consider the channel diameter as well as themethod used to direct the linear crossing of the polymer e.g., anelectric field. The diameter of the channels should correspond well withthat of the labeled polymer. The theory for linear crossing is that thediameter of the channels correspond well with that of the polymer. Forexample the ring-like sliding clamps of DNA polymerases have internaldiameters that correspond well with the diameter of double-stranded DNAand are successful at achieving linear crossing of a DNA molecule. Manykilobases of DNA can be threaded through the sliding clamps. Severalreferences also have demonstrated that linear crossing of DNA throughchannels occurs when the diameter of the channels corresponds well withthat of the diameter of the DNA. (Bustamante, 1991; Gurrieri et al.,1990; Matsumoto et al., 1981).

Single-stranded DNA, as used in the experiment, has a diameter of˜1.6-nm. A channel having an internal diameter of approximately 1.7-3 nmis sufficient to allow linear crossing of a single strand DNA molecule.The diameters of the channel and the DNA need not match exactly but itis preferred that they be similar. For double-stranded DNA which has adiameter of 3.4-nm, channel sizes between 3.5-nm and 4.5-nm aresufficient to allow linear crossing.

As discussed earlier many methods may be used to move the polymerlinearly across the channel and past the interaction station or signalgeneration station. A preferred method according to the inventionutilizes an electric field. An electric field can be used to pull apolymer through a channel because the polymer becomes stretched andaligned in the direction of the applied field as has previously beendemonstrated in several studies (Bustamante, 1991; Gurrieri et al.,1990; Matsumoto et al., 1981). The most related experiments regardinglinear crossing of polymers through channels arise from experiments inwhich polymeric molecules are pulled through protein channels withelectric fields as described in Kasianowicz et al., 1996 and Bezrukov etal., 1994, each of which is hereby incorporated by reference. A briefdescription of these experiments is presented below in order toillustrate one method for enabling linear crossing of polymers.

In a study entitled, “Characterization of individual polynucleotidemolecules using a membrane channel,” Kasianowicz et al., 1996demonstrate the linear crossing of DNA molecules through proteinchannels in a lipid bilayer with an electric field (also described inPCT Published Patent Application WO 96/29593). An excerpt of theabstract follows.

-   -   We show that an electric field can drive single-stranded RNA and        DNA molecules through a 2.6-nm diameter ion channel in a lipid        bilayer membrane. Because the channel diameter can accommodate        only a single strand of RNA or DNA, each polymer traverses the        membrane as an extended chain that partially blocks the channel.

The assay is performed using Staphylococcus aureus-hemolysin as themembrane channel. It has a diameter of 2.6-nm and can remain open forextended periods of time, allowing continuous ionic current to flowacross a lipid bilayer. The hypothesis is that while a DNA moleculetraverses a channel, there should be blockage in the ionic flow. Usingsingle channel recordings, the blockage time is recorded. The length ofblockage time corresponds to the length of the single-stranded DNAmolecule passing through the channel if linear crossing occurs.Initially a potential of −120 mV was applied across the membrane,causing a current flow. When single-stranded DNA was added, twoconsecutive conductance blockades of 300 μs and 1300 μs, respectivelyoccurred.

The data supports two hypotheses: 1) the length of blockage time(denoted as “lifetime”) is directly proportional to the length of theDNA and 2) greater applied voltage shortens the blockage time for agiven length of DNA., indicating that linear crossing indeed occurred.As a control, it was demonstrated that double-stranded DNA do not crossthe protein channels.

Bezrukov et al., 1994 have done similar studies with alamethicin poresin a paper entitled, “Counting polymers moving through a single ionchannel.” Alamethicin pores have internal diameters of 2 nm. Theduration of the conductance blocks were proportional to the length ofthe polymer pulled through the pore, supporting the hypothesis forlinear crossing. The results from Kasianowicz et al., 1996 and Bezrukovet al., 1994 demonstrate that an electric field can drive DNA across aprotein pore channel in a linear fashion.

An experiment was performed to demonstrate according to the inventionthat DNA can pass though fabricated nanochannels. The results of theexperiment are shown in FIG. 11. DNA in various forms was exposed tonanochannels to determine if it could pass through. Only double strandedlinear DNA of 50 kb could pass through an array of 4 nm nanochannels.Folded DNA as modeled by a circular plasmid of the same size, cannotpass through the array. Lanes 1 and 2 are the controls. Circular DNAmigrates slower than linear DNA as expected. Lanes 3 and 4 demonstratethat only linear DNA can pass through the nanochannel array. Sincefolded DNA which has a solvation diameter of approximately 5 nm, cannotpass through the array, the only means by which the linear DNA passesthrough the plate is in a linear fashion.

Using the system described above by Kasianowicz et al., a randomlylabeled polymer, such as DNA can be analyzed by passing the polymerthrough the channel and making unit specific measurements. A channel canbe prepared as a protein pore in a lipid membrane such as that describedin Kasianowicz et al., and also in PCT published patent applicationWO/96/29593. Briefly, the S. aureus protein α-hemolysin is added to thecys side of a lipid bilayer. Lipid bilayers can be formed from, forexample, diphytanoyl phosphatidylcholine by layering the solution on 0.2mm holes in a Teflon film separating two compartments containing buffersolution. After the hemolysin is added, voltage can be applied acrossthe bilayer and varied from 0 mV to 140 mV. DNA which is randomlylabeled is added to the buffer on the cys side of the protein. A voltageis applied which causes the labeled DNA to traverse from this cys to thetrans side of the channel, which has a positive charge. As each unitpasses through the channel, a change in conductance as a result of theblockage of the channel occurs. The change in conductance is dependentupon the size, shape and charge of the unit passing through the channel.If the unit is labeled, the conductance change will reflect theproperties of the label. In this manner, labeled unit can be identified.This method can be used to identify a particular unit or to identifyspecific order of units or distance between units or simply the numberof units which are labeled.

Retrograde movement of the DNA is unlikely because of differences infrictional coefficients for inside the nanochannel and outside. Thepredicted van der Walls interaction between the solvated labeled DNA andthe inside of the nanochannel creates a higher frictional resistance forthe portion of the DNA located inside the nanochannel than that locatedin free solution. This is evidenced by the slower migration of DNA inthe presence of a nanochannel plate (compare lanes 1 and 3 in FIG. 6).The differential in frictional resistance is likely to create a ratchetmechanism in the direct of the desired DNA movement.

Another method for moving a polymer linearly past an interaction stationor a signal generation station involves the use of a molecular motor. Amolecular motor is a device which physically interacts with the polymerand pulls the polymer past the station. Molecular motors include but arenot limited to DNA polymerases and helicases. DNA polymerases have beendemonstrated to function as efficient molecular motors. Preferably theinternal diameters of the regions of the polymerase which clamp onto theDNA is similar to that of double stranded DNA. Furthermore, largeamounts of DNA can be able to be threaded through the clamp in a linearfashion.

The overall structure of the β-subunit of DNA polymerase III holoenzymeis 80 Å in diameter with an internal diameter of ˜35 Å. In comparison, afull turn of duplex B-form DNA is ˜34 Å. The beta subunit fits aroundthe DNA, in a mechanism referred to as a sliding clamp mechanism, tomediate the processive motion of the holoenzyme during DNA replication.It is well understood that the b-subunit encircles DNA duringreplication to confer processivity to the holoenzyme (Bloom et al.,1996; Fu et al., 1996; Griep, 1995; Herendeen and Kelly, 1996; Naktiniset al., 1996; Paz-Elizur et al., 1996; Skaliter et al., 1996). Becausethe sliding clamp is the mechanism of processivity for a polymerase, itnecessarily means that large amounts of DNA are threaded through theclamp in a linear fashion. Several kilobases are threaded through theclamp at one time (Kornberg and Baker, 1991).

Methods for preparing the wall material of the invention are alsoencompassed by the invention. One method for preparing a wall materialaccording to the invention includes the step of covalently bonding theagent to a plurality of discrete locations of a wall material. The agentis bonded to discrete locations on the wall material which are closeenough to an interaction station, such that when an individual unit of apolymer, which is interactive with the agent to produce a signal, ispositioned at the interaction station, the agent interacts with theindividual unit to produce the signal. The discrete locations may be onthe surface of the wall material or may be within the wall material suchthat the agent is embedded in the wall material.

Another method is for attaching a chemical substance selectively at arim of a channel through a wall material that is opaque. An “opaque”material as used herein is a material which is light impermeable at aselected wavelength.

The wall material is provided with photoprotective chemical groupsattached at the rim of the channel through the wall material. Light isthen applied to the photoprotective chemical groups to dephotoprotectthe chemical groups, and a chemical substance is attached to thedephotoprotected chemical groups.

A “photoprotective chemical compound” as used herein is a lightsensitive compound which is capable of becoming chemically reactive whenexposed to light. When light is applied to the photoprotective chemicalgroups the groups become dephotoprotected and are susceptible tointeractions with chemical substances such as light emitting compoundsand quenching compounds.

Localized regions of agent can still be prepared even if thephotoprotective chemical group covers the entire surface of the wall.The light may selectively be applied to regions of the wall on which itis desirable to have the agent localized. For instance the light may beapplied only to the region of the wall surrounding the channel openingsso that only those regions are dephotoprotected. When the agent is addedit will only attach to the wall around the regions surrounding thechannel openings.

Additionally a method for preparing a wall material having localizedareas of light emission on a surface of the wall material is provided. Alight emissive compound is applied to the surface to produce at leastlocalized areas of light emission on the surface. The light emissivecompound may be added directly to the surface of the wall material or itmay be attached indirectly to the surface through a photoprotectivechemical group which has been attached to the surface anddephotoprotected by light.

A “localized area of light emission” as used herein is a region ofconcentrated light emissive compound on the surface of the wall materialwhich defines a target region around a rim of a channel through the wallmaterial for detecting light emission. The localized area can beproduced in several ways. Firstly, the light emissive compound may beattached directly to the wall surface only around the rim of thechannel. Secondly, the light emissive compound may be attached to aphotoprotective chemical group which has been attached selectively tolocalized areas around the rim of the channels. Alternatively thephotoprotective chemical groups may be added to the entire surface or torandom regions of the surface of the wall material but only selectregions are dephotoprotected by light to create localized regions towhich the light emissive compound can be attached. Both thephotoprotective chemical groups and the light emissive compound can beadded to the entire surface or to random regions of the surface of thewall material when a mask is used to create localized regions of lightemission. A mask having openings may be positioned over the wall surfacesuch that the openings in the mask expose localized regions of the wallsurface specifically around the openings of the channels.

A wall material having localized areas of light emission on a surface ofthe wall material may also be prepared by first applying a lightemissive compound to the surface to produce at least localized areas oflight emission on the surface and then by creating a channel in the wallmaterial wherein a rim of the channel forms a target region within thelocalized areas of light emission.

An non-limiting example of a method for constructing an article ofmanufacture having a concentric ring of donor fluorophores around eachchannel is provided to demonstrate a preferred method of the invention.The method is achieved by the use of photolabile protecting groups andlight diffraction. A light impermeable polycarbonate porous medium iscoated on one side with a dense layer of covalent linkers. The linkersare protected by photolabile protecting groups. Light is transmittedthrough the back side of the light impermeable polycarbonate. Since thewavelength of the deprotecting light source (400-500 nm) is much greaterthan the size of the channels (5 nm), each channel acts as a pointsource of light. The intensity of transmitted light is greatest closestto the channel. Accordingly, only those groups close to the channels aredeprotected. Deprotected groups are free to react with donorfluorophores in subsequent chemical reactions to generate concentricrings.

The radius of the concentric ring of deprotection is controlled throughunderstanding the diffractive nature of light. The long wavelength oflight and the small size of the channels set up a situation of verystrong diffraction where diffracted angles are greater than 90°. Lightdoes not pass through the light impermeable polycarbonate surface, butrather is forced to undergo diffraction through the channels. Accordingto Huygen's principle, each channel acts as a secondary point source oflight, spherically radiating. Huygen's principle states that all pointson a wavefront can be considered as point sources for the production ofspherical secondary wavelets. After a time t the new position of thewavefront will be that surface of tangency to these secondary wavelets.A direct consequence of Huygen's principle is the generation ofsecondary point sources at the exit ends of the channels. A furtherconsequence is that the intensity of excitation light decreases withincreasing distance from the center of each channel.

The amount of light exiting the channel and the resulting sphericaldistribution of light intensity can be calculated. The power of lightexiting the channel is given as the cross-sectional area of the channelmultiplied by the intensity of the incident light (I_(o)).

P=I _(o) πR ² _(channel)

Upon exit of the channel, the light becomes a spherically radiatingpoint source. In this particular case, the light source is restricted toradiate to half a sphere (a surface area of 4 πr²/2). The newintensities (I(r)) as a function of the radial distance from the centerof the channel is given as the power of light exiting the channelsdivided by the surface area of radiation at a given distance (r).

$I = {\frac{P}{A} = \frac{I_{o}\pi \; R_{channel}^{2}}{2r}}$

For a 60 W light source 1 cm away from the back side of the lightimpermeable polycarbonate, the intensity decreases from 4.77 W/cm² to0.43 W/cm² at a distance 35 nm (R_(equal)) from the center of thechannel, corresponding to a 91% intensity change. From the aboveequation, the radius of light around each channel can be preciselycontrolled by the intensity of the light source.

Photosensitive protecting groups have been described in great detail(Pillai, 1980). Depending on the type of covalent linker (amine,hydroxyl, carboxylic acid, ketone, sulfhydryl, etc.), the correspondingphotosensitive protecting group is available. For example, amino groupscan be protected with nitroveratryloxycarbonyl (NVOC),2-nitrobenzyloxycarbonyl, and α-substituted 2-nitrobenzyloxycarbony-1groups. The latter two can also be used in the protection of carboxylicacids and hydroxyl groups. Photolytic deblocking of2-nitrobenzyloxycarbonyl derivatives is straightforward, requiring onlya 350 nm lamp and ethanol. The time of deprotection is controlled from 1to 24 hours.

The invention also encompasses an apparatus for detecting a signal. Theapparatus provides a support for the article of manufacture and a sensorfor detecting the signals generated by the interaction which occurs asthe polymer traverses the interaction station. The apparatus includes ahousing with a buffer chamber, a wall defining a portion of the bufferchamber, and having a plurality of openings for aligning polymers, and asensor fixed relative to the housing, the sensor distinguishing thesignals emitted at each opening from the signals emitted at the other ofthe openings to generate opening dependent sensor signals.

An “opening dependent sensor signal” is a signal which arises at anopening in the wall material as a result of an interaction between apolymer and the station.

The wall within the housing defines at least one and preferably twobuffer chambers. A “buffer chamber” as used herein is area which iscapable of supporting a liquid medium. The two buffer chambers may be influid communications with one another.

The wall has a plurality of openings formed by channels within the wall.A “plurality of openings” within the wall means at least two openingsformed by at least two channels. Preferably a plurality of openings isat least 50 openings.

A “sensor” as used herein is a device which responds to a physicalstimulus and transmits a resulting impulse in the form of a signal.Sensors include but are not limited to optical sensors, temperaturesensors, pressure sensors, auditory sensors, magnetic sensors,electrical, mechanical, radioactive and motion sensors. Preferably thesensor is an optical sensor. As used herein an “optical sensor” is adevice which detects and converts input electromagnetic radiationsignals an impulse. The impulse can be measured and stored as data.Optical sensors are well known in the art and include microscopes. Thesensor is fixed relative to the housing such that the sensor is capableof detecting signals generated at the interaction station. It is notnecessary that the sensor be secured or attached directly to thehousing.

A microprocessor as used herein is a device for collecting and storingsensor signals. In general a microprocessor is a chip containing severalelectronic components such as ROM, RAM, registers, and I/O controls.Conventional microprocessors are well known in the electronics arts.

An example of an apparatus constructed to hold a nanochannel (ormicrochannel) plate (120) which is capable of generating an electricfield is presented in FIG. 12. The electric field, created by electrodes(128, 130), is used to draw the DNA through the nanochannels. Theexemplary nanochannel plate is immersed in a slightly viscous buffersolution which helps to slow the transit of the polymer through thenanochannel, so as to allow for a longer signal collection time perbase. In addition, on either side of the plate are electrodes (128, 130)immersed in the buffer solution. The ensemble of nanochannel plate,buffer compartments (122), and electrodes are contained in an opticalquality glass chamber suitable for image analysis and are positionedadjacent to a 60×1.4 NA oil objective (126).

As discussed above the use of an electric field to cause the polymer tomove linearly through a channel is preferred. The use of an electricfield is suitable because the stretched, linear orientation of a polymerin an electric field is favorable for linear crossing of nanochannels.Furthermore, the rate of polymer movement can be controlled by voltage.Lastly, an electric field does not adversely affect FRET.

Light microscopy (Bustamante, 1991; Gurrieri et al., 1990; Matsumoto etal., 1981, Rampino and Chrambach, 1990; Schwartz and Koval, 1989; Smithet al., 1989), linear dichroism (LD) (Akerman et al., 1990; Akerman etal., 1985, Moore et al., 1986), fluorescence-detected LD (Holzwarth etal., 1987), and linear birefringence (Sturm and Weill, 1989; Chu et al.,1990) can be used to study the instantaneous changes in shape of DNAmolecules undergoing gel electrophoresis. In these studies DNA is shownto be strongly oriented and stretched.

Guirrieri et al., 1990 has demonstrated linear and stretchedconformation of DNA molecules in an electric field. In each of thecases, the DNA molecule is clearly aligned in the direction of theapplied electric field. The method used to visualize the DNA moleculescombines fluorescent DNA labeling and use of an image intensifier-videocamera system (Bustamante, 1991; Houseal et al., 1989; Morikawa andYanagida, 1981; Matsumoto et al., 1989; Yanagida et al., 1983). The DNAmolecules shown are T2 molecules which are 164 kbp long.

The orientation of DNA in an electric field has been well studied withlinear dichroism and electric dichroism (Ding et al., 1972; Yamaoka andChamey, 1973; Colson et al., 1974; Hogan et al., 1978; Priore and Allen,1979; Yamaoka and Matsuda, 1981; Wu et al., 1981). In fact, the firststudies done on DNA orientation have been performed with these twotechniques. DNA was first studied in solution and then subsequently inelectrophoretic gels. Studies both in solution and in gels yield similarresults in that the DNA molecules are indeed oriented in the directionof the electric field.

Native DNA exhibits a negative UV linear dichroism (LD) as a result ofthe preferential orientation of the nucleotide bases nearlyperpendicular to the field. The orientation of DNA has been attributedto the presence of either a permanent or an induced dipole moment. Forexample, Ding et al., 1972 describe their observation of DNA in aTris-cacodylate buffer diluted in ethanol to give 80% v/v ethanol.

LD data from single electric pulse of 9 V/cm. Duration of pulse isindicated by the horizontal bar. At steady-state, the plateau of theLD^(r) reads close to −1.5, which means that the DNA is oriented in thedirection of the electric field (Akerman et al., 1990).

Akerman et al., 1990 have performed LD studies on pulsed-field gelelectrophoresis and similar results are obtained as in solution. Akermanprovides a plot of LD^(r) versus time. LD^(r) represents orientation inthe electric field. A value of −1.5 means that the DNA molecule isoriented in the electric field. A horizontal bar on the x-axis denotesthe duration of the electric pulse. The DNA is in 1% agarose. A shorttime after the beginning of the pulse, the curve plateaus at a LD^(r)close to −1.5, meaning that the DNA is oriented a short time after thebeginning of the pulse. When the pulse is turned off, the DNA no longerorients itself in the field and the LD^(r) curve no longer remains at−1.5.

The rationale for DNA orientation in electric fields is based on eithera permanent or induced dipole moment in DNA. Recall that dipoles alignthemselves in the direction of electric fields to minimize torque. Hoganet al., 1978 have proposed a rationale for the induction of a dipolemoment in DNA that relates to anisotropic ion flow:

-   -   In order to explain the dependence of the dichroism on the        electric field, the ionic strength of the medium, and the length        of the macromolecule, we propose a new model in which        anisotropic ion flow produces an asymmetric ion atmosphere        around the polyelectrolyte, resulting in an orienting torque.

The conclusion from the discussion on DNA alignment in electric fieldsis that DNA molecules and other polymers align themselves in thedirection of electric fields whether in an electrophoretic gel or insolution. Solution studies were performed before electrophoreticstudies. The implications of DNA alignment in an electric field furthersupport the fact that DNA molecules and other polymers can be drivenacross nanochannels in a linear fashion. The unfolded orientation of theDNA is also important in the linear crossing of DNA molecules. Asdescribed above DNA and other polymeric molecules can be driven throughprotein channels in a linear fashion with an electric field. The datadescribed herein rationalize this further and give a theoretical basisto why DNA molecules can behave in such a fashion. Given that DNA hasbeen shown to pass through protein pores in a linear fashion and thatDNA molecules align themselves in the direction of electric fields,linear crossing of DNA through nanochannels can be achieved.

The rate at which a polymer moves is also important because the durationof energy transfer is important. The longer an acceptor remains in thedonor fluorophore layer, the greater the signal generated. Millisecondintegration times allow for unequivocal signal detection. Since thelayer of donor fluorophore is roughly 40 Å, the rate of polymer movementthat is needed can be approximated as 40 Å/10 ms, or 4000 Å/s. Innanochannel FRET sequencing, the proposed rate is achieved bycontrolling either the voltage of the applied electric field or thefrictional coefficient of the polymer molecule.

The rate of DNA movement can be determined given the equation thatdefines the movement of a DNA strand in an electric field (Tinoco etal., 1995):

$u = \frac{ZeE}{f}$

where Z is the (unitless) numbers of charges, e=1.6022×10⁻¹⁹ coulombs, Eis the electric field in colts per m, and f is the frictionalcoefficient in kg/s. The velocity of motion this depends on themagnitude of the electric field E, the net charge of the molecule, andthe size and shape of the molecule as characterized by its frictionalcoefficient, f. The net charge on the molecule is designated by Ze. Thefrictional coefficient can be determined from the following equation(Tinoco et al., 1995):

f=kf _(o) =k(6πηr)

where h is the viscosity coefficient, r is the radius for a spherehaving the same volume as the DNA, k is a shape factor (which for astrand of DNA is about 1.7), and f_(o) is the frictional coefficient fora sphere having the same volume as the DNA.

Rationally, it can be seen that either the frictional coefficient or themagnitude of the electric field can be used to control the rate ofpolymer movement. For different voltages in a given system, there aredifferent polymer migration rates. In a similar fashion, largermolecules or molecules in more viscous media have slower molecularmobility. From examples in gel electrophoresis, calculations of theeffects of electric field strength, polymer size, and frictionalcoefficient are possible. It is important to understand a viscous mediumcan achieve the same desired effects on the frictional coefficient as agel. Since the frictional coefficient is inversely proportional to theelectrophoretic rate, doubling viscosity decreases electrophoretic rateby two-fold.

Electrophoresis in viscous solvents has been performed by a number ofgroups (Chang and Yeung, 1995; Bello et al., 1994; Jumppanen andRiekkoloa, 1995; Sahota and Khaledi, 1994; Klenchin et al., 1991; Harrisand Kell, 1985; Korchemnaya et al., 1978). Sahota and Khaledi, 1994demonstrate electrophoresis in formamide, which has a viscosity of 3.3cP at 25° C., triple that of water (0.89 cP, 25° C.). Electrophoresis informamide is more favorable than in water because formamide has a higherdielectric constant than water and can solubilize many supportingelectrolytes. It is commonly used in mixtures with water inelectrophoretic buffers. Mixed glycerol solutions,N-methyl-2-pyrrolidone, acetic acid, and N-methyl formamide have alsobeen used (Korchemnaya et al., 1978). A large range of viscosities arepossible, ranging from 1 cP to as high as 25 cP (Bellow et al., 1994).The actual viscosity of the solution needed depends on the frictionalcoefficient of polymer molecules in nanochannels. The net frictionalcoefficient thus depends on viscosity and also the frictional effects ofthe nanochannels on the polymer. From the small bore of the nanochannel,there are frictional effects from solvent trapping between the walls ofthe nanochannels and the labeled polymer. The magnitude of these solventtrapping effects is similar to those arising from gel electrophoresis.As a result, only a slightly viscous solution may enhance the desiredrate of polymer movement.

For a given electric field, the electrophoretic mobility for differentsized molecules can be determined by knowing the relationship betweenmolecular weight and distance migrated, given as:

Log M=a−bx

where M is the molecular weight of the nucleic acid and x is thedistance migrated (proportional to mobility). a and b are constants fora given electric field.

To summarize, the rate of polymer movement in an electric field can becontrolled by the electric field, frictional coefficient, and molecularsize. Desired values for the rate of polymer movement are readilyachievable.

There are no adverse effects of an electric field on FRET. Since energytransfer is related to short range electrical dipole interactions, thepresence of an outside electric field may negatively affect the abilityof donor-acceptor pairs to undergo FRET. The presence of an externalelectric field has no adverse effects on FRET as seen from recentstudies performed with FRET primers and gel-based automated DNAsequencing (Glazer and Mathies, 1997; Hung et al., 1996; Ju et al.,1996a; Ju et al., 1996b; Marra et al., 1996; Wang et al., 1996; Wang etal., 1995). FRET primers are used in the labeling of sequencing reactionproducts because of the large Stoke's shift and thus greater fluorophorediscrimination. In these experiments, the FRET labeled DNA fragments arerun on a gel and concurrently, in the presence of the electric field,the fragments are detected with a CCD camera and laser. Either afour-color capillary electrophoresis system or Applied Biosystems 373Asequencer is used. These experiments demonstrate that energy transferoccurs in the presence of a strong electric field.

An exemplary design of a nanochannel apparatus is shown in FIG. 13,which consists of two fused Pyrex cells (132, 134) that hold thenanochannel plate (140). The upper (136) and lower (138) buffer regionsare contained in the smaller (134) and larger (132) Pyrex cells,respectively. The buffer regions are sealed off with glass coverslips(146) that have been modified with indium tin oxide (ITO). Indium tinoxide is a clear conductor which functions in this system as electrodes(142). The electrodes lead to a variable voltage supply (152) consistingof a load resistance R_(L) (one to several G, to approach a constantcurrent system) and a rheostat-controlled voltage from a voltage source.A voltmeter is connected in parallel. Under the glass coverslip an oil(144) coated immersion objective (150) is positioned to detectfluorescence signals (148).

The polymer is loaded into the upper buffer region and the electricfield is used to drive the polymer to the lower chamber. The buffer hasthe desired viscosity and electrolytic properties. Epiillumination andsignal collection are possible through the same 60×, 1.4 NA oilimmersion objective. The dimensions of the apparatus are similar to thatof a conventional microscope slide (75 mm×25 mm) for ease of mounting.The gap between the nanochannel plate and the glass coverslip is 0.4 mm.A thin coverslip with a thickness of 0.1 mm is shown. These dimensionsare preferred because long working distance objectives are available.For instance, the CFI₆₀ system from Nikon has 60×, 1.4 NA objectivesthat can examine specimens over 0.6 mm thick.

A temperature control block is used to prevent Joule heating and thermalfluctuations. The block can surround the nanochannel apparatus and hasinlet/outlet ports for coolant flow. The block is hollow to allowuniform temperature control of the nanochannel apparatus. There areinlet/outlet ports on either side of the apparatus that allow therecycling of the coolant through the hollow chamber.

Several groups have used specialized apparatuses for fluorescencemicroscopy observations of electrophoresis (Rampino and Chrambach, 1991;Matsumoto et al., 1981; Smith et al., 1989; Bustamante, 1991; Gurrieriet al., 1990; Schwartz and Koval, 1989; Smith et al., 1990). Theapparatus used by Smith et al., 1990 for the observation of DNAmolecules undergoing pulsed-field gel electrophoresis is described:

-   -   A 20-0 drop of molten agarose was placed between two microscope        coverslips (24 mm×24 mm) and allowed to cool for 10 minutes.        This sandwich arrangement was placed on top of a regular        microscope slide and sealed at the four corners with fingernail        polish. The slide had been previously prepared with four copper        electrodes fixed on it with epoxy adhesive. This apparatus was        then refrigerated at about 5° C. for 30 minutes. Molten agarose        was dropped over the electrodes to complete the electrical        connection, and TBE buffer (0.5×) was occasionally added to keep        it wet.

Rampino and Chrambach, 1991 describe a more complex apparatus that haslarger buffer regions, platinum strip electrodes, and a region forcoolant flow. The entire apparatus is mountable on the microscope stage.The actual gel itself is sandwiched between two glass coverslips andplaced in the center of the apparatus, in contact with buffer blocks oneither side. The design allows for a homogenous electric field andtemperature control. In contrast to the apparatus described above, theRampino and Chrambach apparatus is designed for standard and notpulsed-field gel electrophoresis.

Briefly, a description of how the apparatus (shown in FIG. 13) of theinvention functions in relation to polymer is described below. A polymersuch as DNA is labeled (intrinsically or extrinsically) by the methodsof the invention or any other method known in the art. The labeled DNAis placed in a buffer solution, which is preferably slightly viscous.The buffer is added to the upper buffer chamber 136. An electric fieldis created using the electrodes and the DNA is caused to enter thenanochannels of the nanochannel plate (140) in a linear manner. As thelabeled DNA emerges from the other side of the nanochannel plate thelabel is caused to interact with an agent or the environment surroundingthe channel to produce a signal or polymer dependent impulse which isdetected by the detection device. The detected signal or polymerdependent impulse is stored and processed to reveal structuralinformation about the polymer.

A final aspect of the invention encompasses a method for preparingnucleic acids for use according to the methods of the invention as wellas for any other use in which it is desirable to utilize randomlylabeled nucleic acids. The method involves the steps of contacting adividing cell with a nucleotide analog, isolating from the cell nucleicacids that have incorporated the nucleotide analog, and modifying thenucleic acid with incorporated nucleotide analog by labeling theincorporated nucleotide analog. A “nucleotide analog” as used herein isa molecule which can be substituted for A, T, G or C but which has amodified structure. Nucleotide analogs include for example but are notlimited to a brominated analog, 2,4-dithiouracil, 2,4-Diselenouracil,hypoxanthine, mercaptopurine, 2-aminopurine, and selenopurine. Theincorporated nucleotide analog may be labeled with an agent selectedfrom the group consisting of a light emitting compound, a quenchingsource and a fluorescence excitation source.

A dividing cell is “contacted” with a nucleotide analog by any meansknown in the art for incorporating nucleotides into living cells. Forinstance the nucleotide analog may be added to the cell culture mediumand taken up by the cell naturally. In order to optimize the uptake ofthe nucleotide analog the dividing cell may be growth arrested usingconventional means prior to adding the nucleotide analog to the mediumand then the arrest removed to allow the cells to reenter the celldivision cycle once the nucleotide analog is added to the medium. Thenucleic acids having the incorporated nucleotide analog may then beisolated after the cells have reentered and completed at least one celldivision cycle.

An example of a method for incorporating a nucleotide analog into DNA isprovided in Bick and Davidson, Proc. Nat. Acad. Sci., 71:2082-2086(1974). Bick and Davidson grew the BrdU-dependent cell line, B4, in abasic growth medium of Dulbecco's modified Eagle's medium supplementedwith 10% fetal-calf serum (E medium) containing 0.1 mM BrdU. 100-mmFalcon plastic tissue culture dishes were inoculated with 10⁶ B4 cellsin E medium containing 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 10μM BrdU (E-HAB medium) and the cells were passaged three times at highdensity (5×10⁵ cells per 100-mm dish) in E-HAB medium over six weeks.The cells were then plated at low density (1000 cells per 6-mm dish) inE-HAB medium and three weeks later, approximately 10 large colonies wereobserved in each dish. The cells from one dish were harvested andmaintained as a new cell line called HAB. Because of the expectedphotosensitivity of BrdU-containing cells, the HAB cells were at alltimes protected from environmental lighting. After 100 cell generationsin E-HAB medium, an aliquot of HAB cells was transferred back to Emedium. A new subline of cells, called HAB-E, was isolated andmaintained in E medium.

Bick and Davidson grew the cell in the media (described above) to whichwas added H₃ ³²PO₄ (New England Nuclear Corp.) at an activity of 2μCi/ml. After 2-3 population doublings the cells were harvested and DNAwas isolated as described in Davidson and Bick (Proc. Nat. Acad. Sci.,70:138-142 (1973)) except that after the first phenol extraction, DNApreparations were routinely treated with RNase A at a concentration of50 μg/ml for 60 min at 37°. After a second phenol extraction, the DNAwas extensively dialyzed against 10 mM Tris-HCl, 10 mM pH 7.6, 1 mMEDTA, and then finally dialyzed against 10 mM Tris-HCl pH 7.6, 1 mMEDTA. Other methods for isolating DNA are well known in the art. Seee.g., Sambrook. et al., Molecular Cloning: A Laboratory Manual, 2ndedition, Cold Spring Harbor Laboratory Press, 1989).

The method of the invention for preparing nucleic acids is referred tohereinafter as in vitro base specific labeling (IBSA). IBSA involves theculture of cells derived from a subject in the presence of nucleotideanalogs. The nucleotide analogs are either capable of subsequentchemical modifications, have a high molar extinction coefficient in thedonor emission wavelengths, or have linkers for the attachment ofacceptor labels. The most important analogs are the ones which containfunctional groups that do not interfere with base pairing during DNAreplication. The 8-substituted purines, 5-substituted pyrimidines, and6-substituted pyrimidines satisfy this criteria.

A non-limiting example of an IBSA scheme is set forth in detail below.The method is outlined in FIG. 14 and covers the steps from initial DNAisolation to genomic labeling and to final sequence analysis. Cells areisolated from the subject and then grown. At a time when there areenough cells to provide adequate genetic material, the cells arearrested in the cell cycle. The arrest can occur at any point in thecycle as long as it does not interrupt the S phase. Nucleotide analogsthat can be easily chemically derivitized, such as8-bromo-2′-deoxypurines and 5-bromo-2′-deoxypyrimidines, are added tothe growth medium. The cells are then grown for one cycle and thegenomic DNA from the cells are isolated. The resulting DNA is a chimeraof labeled and unlabeled DNA, a product of semiconservative replication.The advantage in having the DNA with only one strand labeled is so thatthe resultant duplex DNA can be used in a sequencing method of theinvention.

The genomic DNA is broken down into sizes of approximately 100 kb. Thegenomic DNA is chemically labeled and end-labeled at this point. Theadvantage of culturing cells in brominated analogs is that such amodification activates the bases for further modification, the additionof acceptor labels to the bases. For a human genome, there areapproximately thirty thousand fragments. The fragments are analyzedsimultaneously by the sequencing methods of the invention. Fragments ofsimilar size and potential sequences are grouped together and populationanalyses are performed to generate DNA sequence information. Sequencingof a genome can be completed in approximately six hours. The time fromisolation of a subject's cell to the end of sequencing is approximatelyone week. The following paragraphs describe each individual step indetail.

The first step to the outlined scheme is cell culture and cell cyclearrest. The purpose of the cell cycle arrest is to generate asynchronous population of cells which can undergo DNA replication at thesame time to ensure all the resultant DNA will be a chimera of labeledand unlabeled DNA. There are many methods to synchronize cells includingmethods which arrest cells at either M or S phase. Only an M phase blockwould produce the desired genomic chimeras. Examples of metaphaseinhibitors include nitrous oxide under pressure, vinblastine (Marcus &Robbins, 1963), and colcemid.

Colcemid blocks cells in metaphase but can be reversed by washing(Mitchison, 1971; Kato & Yoshida, 1970; Stubblefield, 1968). Cells aregrown for a few hours in colcemid, resulting in a synchronous culture(Wunderlich & Peyk, 1969). Nitrous oxide under pressure (Rao, 1968) isadvantageous in that it can be easily removed. Removal of the block andaddition of the desired brominated base analog allows for a synchronousentrance into S phase.

The nucleotide analogs which can be used include but are not limited to8-bromo-2′-deoxyadenosine, 8-bromo-2′-deoxyguanosine,5-bromo-2′-deoxycytidine, and 5-bromo-2′-deoxyuridine. These brominatednucleotide analogs are activated for coupling to nucleophiles (Traincardet al., 1983; Sakamoto et al., 1987; Keller et al., 1988; Hermanson,1996). Coupling of amine-containing acceptor labels and fluorophores tothe base analogs is done at ambient temperatures (35° C.). The finalpositions of the acceptor labels are such that the base-pairing of thelabeled fragment is not affected.

Brominated nucleotide analogs are commercially available (Sigma Corp.,Fluka Inc., Fisher Scientific, Inc.) or can be prepared by reaction withbrominating reagents. Hermanson, 1996 outlines a protocol forbromination of DNA at thymines, cytidines, and guanosines usingN-bromosuccinimide (NBS). Adenine residues are prepared from othermethods which do not require NBS. For an example,8-bromo-2′-deoxyadenosine and 8-bromo-2′-deoxyguanosine can besynthesized by reaction of the native base with bromine water (Ikeharaet al., 1969a; Ueda et al., 1974; Faerber & Scheit, 1971; Kochetkov etal., 1968). Bromination at the eighth position of the purines can alsoachieved via the nucleotide morpholidate (Lang et al., 1968; Ikehara etal., 1969b). The solvent condition for bromination is especiallyimportant. Bromination of 2′-deoxyadenosine does not take place indimethylformamide at 0° C., but if the reaction is conducted at 50-60°C. in glacial acetic acid, 8-bromo-2′-deoxyadenosine is formedrelatively easily (Gueron et al., 1967; Rahn et al., 1965).

Brominated and other base analogs have been demonstrated to beefficiently incorporated into genomic DNA during cell culture (Balzariniet al., 1984). A particular example is the incorporation of5-bromo-2′-deoxyuridine into cells used for flow cytometry.5-bromo-2′-deoxyuridine is incorporated into newly synthesized DNA inthe place of thymidine (Crissman et al., 1990; Poot & Hoehn, 1990;Bohmer, 1990; Gaines et al., 1996; Nicolas et al., 1990). The percentageof substitution of base analog can be very high as according to Bick andDavidson, 1974 in their paper discussed above and titled, “TotalSubstitution of Bromodeoxyuridine for Thymidine in the DNA of aBromodeoxyuridine-Dependent Cell Line. The following is a paragraph fromthis paper:

-   -   To obtain total substitution, BrdU-dependent cells were exoposed        to culture medium containing aminopterin, a powerful inhibitor        of thymidine biosynthesis, and BrdU in the absence of added        thymidine . . . . We report here the isolation of a cell line in        which at least 99.8% of the thymidine in the nuclear DNA has        been replaced by BrdU.

In the current proposed random one-base labeling scheme, percentages ashigh as 99.8% substitution are not necessary but such data gives insightinto possibilities of cellular incorporation of base analogs. Otherbrominated base analogs can be similarly incorporated into mammaliancells with ease (Stewart et al., 1968). A few examples of non-brominebase analogs that have been incorporated in this manner include2-aminopurine (Glickman, 1985), 5-propynyloxy-2′-deoxyuridine, and5-ethynyl-2′-deoxyuridine (Balzarini et al., 1984).

After generation of the appropriately brominated DNA chimeras throughcell culture, the chimeric genomic DNA is prepared for analysis byperforming several steps, including an optional step of chromosomesorting. 100 kb DNA fragments can be generated from the partialdigestion of flow sorted chromosomes. The optional flow sorting step hasbeen covered in detail by many manuals and textbooks (de Jong et al.,1989; Fawcett et al., 1994) and will not be discussed in detail. Inbrief, the cells are arrested in metaphase by the addition of colcemid.Several million of the cultured cells are gently lysed to release themetaphase chromosomes into a suspension buffer in which they are stainedwith one or more fluorescent dyes. The target chromosomes are thenidentified by their pattern of fluorescence emission, using either asingle fluorophore in a one laser system, or two fluorophores in a twolaser detection system. The sorted chromosomes are then prepared andpartially digested as according to protocols described by Glover andHames, 1995 in DNA Cloning 3. Sizes on the order of 100 kb are obtained.

-   -   Fluorophore labeling of the brominated DNA can be done either        before or after partial digestion. The base bromination        activates the bases for nucleophilic displacement. Many        different fluorophores with amine/hydrazine groups can be used        including the popular dyes fluorescein, coumarin, eosin,        rhodamine, and Texas Red (Molecular Probes, Oreg.). These have        absorptions and emissions in the visible region. The amine group        on these fluorophores attack the 8 position of the purines and        the 5 position of the pyrimidines, undergoing a nucleophilic        displacement reaction. The experimental protocol for the        displacement reaction is described by Hermanson, 1996. A        modified outline of the labeling scheme described is given        below.

Protocol for Labeling Brominated DNA

-   -   1. Dissolve a hydrazine or amine-coupled fluorophore in water at        a concentration of 80-100 mM.    -   2. Add 25 μl of the fluorophore solution to a bromine-activated        DNA solution.    -   3. React for 1 h at 50° C.    -   4. Purify by ethanol precipitation. Add 20 μl of 4 M LiCl and        500 μl of ethanol (chilled to −20° C.). Mix well.    -   5. Store at −20° C. for 30 min, then separate the precipitated        DNA by centrifugation at 12,000 g.    -   6. Remove the supernatant and wash the pellet with 70% and 100%        ethanol, centrifuging after each wash.        Redissolve the labeled DNA pellet in water and store at −20° C.

From the genomic partial digestion, approximately thirty thousand 100 kbfragments are created. The fragments are end-labeled either withterminal transferase or through ligation to short labeledpolynucleotides. The fluorescent labels incorporated by end-labeling areuniquely identified either by different spectral absorbances or aspecific sequence of labels (as in the ligation scheme). Afterend-labeling, the prepared DNA is subject to nanochannel FRET sequencingas described above. The data is sorted by fragment size and potentialsequence. Through population analysis, the DNA sequence is generated.

FIG. 15 presents the general scheme for deciphering DNA sequence fromIBSA labeling. A sample of genomic DNA has two of its bases labeled (inthis case A and C). The end-labeled and internally labeled DNA fragmentsare analyzed by FRET sequencing. The resulting data is sorted byfragment size and potential sequence. Potential sequence is defined asthe pattern of sequence-specific FRET signals generated for a particularfragment of DNA. A potential sequence does not give sequenceinformation, but rather allows DNA molecules to be uniquely identified.Population analyses are performed to determine all the positions of theA's and C's on each of the fragments. Complementary strands are paired.The process of determining base position is repeated for only one of thebases (A). Comparison of the resulting data for one labeled base and twolabeled bases generates the DNA sequence.

Hundred kilobase fragments are not always desired for sequencing.Expression mapping and single gene sequencing require much shorterlabeled fragments. The generation of shorter labeled fragments can beachieved by nick translation, primer extension, and the polymerase chainreaction (PCR). In addition, smaller fragments can also be analyzed by amodification of the Sanger reaction and the use of terminaltransferases. With enzymatic methods, large nucleotide analogs can oftenbe incorporated (Hermanson, 1996).

In primer extension, modified nucleoside triphosphates are added to aDNA template using a defined amount of the desired primer. The form ofpolymerase used is the Klenow fragment, which lacks the 5′-3′exonuclease activity of intact E. coli DNA polymerase I (Kessler et al.,1990; Feinberg and Vogelstein, 1983, 1984). Equivalent enzymes are theSequenase 2.0 and T7 polymerases. This method is a simple method ofpreparing internally labeled target DNA. Using the method, synthesis oflarge internally labeled DNA fragments with lengths greater than 10kilobases have been synthesized with one of the nucleotides replaced bya modified base. A brief summary of the protocol involves usingSequenase 2.0 (Amersham). The annealing reaction consists of 1 μl primer(0.5 μmol/μl), 2 μl Sequenase buffer (5×), 5 μg plasmid DNA, and 10 μlsterile distilled water. After incubation in a 65° C. waterbath for 2minutes, annealing is allowed to occur at 30° C. for 30 minutes. Theactual reaction mixture consists of 10 μl of annealing reaction(primer-template), 1 μl dithiothreitol (0.1 M), 1 μM of each dNTP, 0.1μM of a fluorophore dNTP, and 0.025 U/μl of Sequenase. After mixing, thereaction is allowed to proceed at room temperature for 15 minutes.

Using primer extension, Ambrose et al. (1993) and Harding and Keller(1992) have demonstrated the synthesis of large fluorescent DNAmolecules. In their experiments, complete replacement of one pyrimidinewith a fluorophore analog was achieved in DNA longer than 7 kb.Fluorescent DNA between 5 to 7 kb in length, in which two pyrimidinesare completely labeled, have also been reported by the same authors.Large fragments using other unusual base analogs such as7-(2-oxoethyl)guanine, 2′-amino-2′-deoxycytidine, 8-oxopurine, andN-4-aminodeoxycytidine have been synthesized as well (Barbin et al.,1985; Purmal et al., 1994; Aurup et al., 1994; Negishi et al., 1988).

Nick translation takes advantage of the ability of Escherichia coli DNApolymerase I to combine the sequential addition of nucleotide residuesto the 3′-hydroxyl terminus of a nick [generated by pancreaticdeoxyribonuclease (Dnase) I] with the elimination of nucleotides fromthe adjacent 5′-phosphoryl terminus (Meinkoth & Wahl, 1987; Rigby etal., 1977; Langer et al., 1981; Holtke et al., 1990). Many modifiedbases have been incorporated by nick translation (Meffert and Dose,1988; Gebeyehu et al., 1987; Gillam and Tener, 1986). DNA polymerase Icauses breaks to be filled as rapidly as they are formed, incorporatingthe desired nucleotides into the original strands. Since a quantity oflabeled nucleoside triphosphates are present during the reaction, thelabels get incorporated and the parent strands are modified. Nicktranslation involves a mixture of the double-stranded DNA target, 1 μlof DNAse I at a concentration of 2 ng/ml, 1 μl each of three types ofunmodified deoxynucleoside triphosphates (dNTPs at 100 μMconcentration), 1 μl of a labeled dNTP (at 300 μM), 32 μl water, and 1μl of DNA polymerase containing 5-10 units of activity. Reactionproceeds for 1 h at 15° C. Reaction is quenched by 4 μl of 0.25 M EDTA,2 μl of 10 mg/ml tRNA, and 150 μl of 10 mM Tris, pH 7.5. Labeled DNA ispurified by ethanol precipitation (Bethesda Research Laboratories, NewEngland Nuclear, and Amersham).

Direct PCR labeling not only incorporates the desired label into thetarget DNA, but also amplifies the amount of labeled DNA (Saiki et al.,1985, 1988). PCR uses the heat-stable forms of DNA polymerase, mostcommonly, the Taq polymerase from Therms aquaticus. Taq polymerase hasthe capability to incorporate labels into growing DNA copies with eachcycle of PCR. In this manner, DNA fragments up to 3 kb can be labeled.Wiemann et al., 1995 describe the method of internally labeling PCRproducts with fluorescein-15-dATP, a protocol that can be applied toother fluorophores. The reaction mixture for PCR incorporation offluorophores consists of 1 μM of each primer, 200 μM of each DNTP, 10 μMof a fluorophore-dNTP, and 0.025 U/μl of Taq polymerase (Perkin-Elmer,Norwalk, Conn.). The reaction buffer consists of 10 mM Tris-HCl, pH 8.3,50 mM KCl, 1.5 mM MgCl₂, and 0.01% (wt/vol) gelatin. PCR is performedusing thirty cycles.

Sequencing reactions generated by the Sanger method can also be analyzedwith the aid of enzymatic labeling. Each chain-terminated fragment islabeled with a 5′ and 3′ fluorophore. The 5′ fluorophore is incorporatedas part of the primer and the 3′ end-label is added by terminaltransferase. Terminal transferase labeling was originally developedusing radiolabeled nucleoside triphosphates (Roychoudhury et al., 1979;Tu and Cohen, 1980). The technique was later applied to non-radioactivenucleotide analogs (Kumar et al., 1988). The technique involves additionof the target DNA to (a) 20 μl of 0.5 M potassium cacodylate, 5 mMCoCl₂, 1 mM DTT, pH 7, (b) 100 μM of a modified deoxynucleosidetriphosphate, 4 μl of 5 mM dCTP, and 100 μl of water. Terminaltransferase is added to a final concentration of 50 units in thereaction mixture and reacted for 45 minutes at 37° C. (Hermanson, 1996).

A post-labeling procedure can be used as well. In this method, a baseanalog with a linker arm for attaching a fluorophore is initiallyincorporated into the DNA using the methods described above. Subsequentattachment of fluorophores to the linker arms is possible using covalentlinking techniques such as those described by Waggoner (1995). Jett etal. (1995) has demonstrated the full length synthesis of M13 DNA (7250bp) with complete incorporation of one of the following:543-aminopropynyl)-dCTP, 5-(3-aminoallyl)-dCTP, or5-(3-aminoallyl)-dUTP. Covalent fluorophore attachment to the linkerswas also shown. The advantage of this technique is that the label neednot consist of individual fluorescent molecules, but could be largermolecules with greater number of dyes. Such examples includephycobiliproteins, dye-filled beads, tagged proteins, or chains offluorescent tags.

Each of the foregoing patents, patent applications and references isherein incorporated by reference in its entirety. Having described thepresently preferred embodiments in accordance with the presentinvention, it is believed that other modifications, variations andchanges will be suggested to those skilled in the art in view of theteachings set forth herein. It is, therefore, to be understood that allsuch variations, modifications, and changes are believed to fall withinthe scope of the present invention as defined by the appended claims.

REFERENCES

-   1. Akerman, B., Jonsson, M., Moore, D., and Schellman, J. 1990.    Conformational dynamics of DNA during gel electrophoresis studied by    linear dichroism spectroscopy. In Electrophoresis of Large DNA    Molecules (Lai, E. and Birren, B. W., Eds). Cold Spring Harbor    Laboratory Press, New York.-   2. Akerman, B. Jonsson, M., and Nordn. 1985. Electrophoretic    orientation of DNA detected by linear dichroism spectroscopy. J.    Chem. Soc. D. Chem. Commun. 422.-   3. Alkens, R. 1992. Properties of low-light level slow-scan    detectors in Fluorescent and Luminescent Probes for Biological    Activity. Mason, W. T. (Ed.) Harcourt Brace & Company, Cambridge,    England.-   4. Allen, M. J., Balooch, M., Subbrah, S., Tench, R. J., Sickhaus,    W., and Balhorn, R. 1991. Scanning Microsc. 5:625.-   5. Ambrose, W. P., Goodwin, P. M., Jett, J. H., Johnson, M. E.,    Martin, J. C., Marrone, B. L, Schecker, J. A., wilkerson, C. W., and    Keller, R. A. 1993. Application of single molecule detection to DNA    sequencing and sizing. Ber. Bunsenges. Phys: Chem. 97:1535-1542.-   6. Andersson-Engels, S., Johannson, J., and Svanberg, S. 1990.    Multicolor fluorescence imaging systems for tissue diagnostics.    Proc. SPIE—Bioimag. Two-Dimens. Spectrosc. 1205:179-89.-   7. Aurup, H., Tuschl, T., Benseler, F., Ludwig, J., and    Eckstein, F. 1994. Oligonucleotide duplexes containing    2′-amino-2′-deoxycytidines: thermal stability and chemical    reactivity. Nucleic Acids Res. 22:20-4.-   8. Arts, E., Kuiken, J., Jager, S., and Hoekstra, D. 1993. Fusion of    artificial membranes with mammalian spermatozoa. Specific    involvement of the equatorial segment after acrosome reaction.    Eur. J. Biochem. 217:1001-9.-   9. Bains, W. 1991. Hybridization methods for DNA sequencing.    Genomics, 11:294-301.-   10. Bains, W. 1997. Hybridization for sequencing of DNA: In Molcular    Biology and Biotechnology, R. A. Meyers, Ed. VCH Publishers, New    York.-   11. Balzarini, J., De Clercq, E., Ayusawa, D., and Seno, T. 1984.    Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell    line as a tool for studying the thymidine salvage pathway and the    incorporation of thymidine analogues into host cell DNA. Biochem. J.    217245-52.-   12. Barbin, A., Laib, R. J., and Bartsch, H.1985. Lack of miscoding    properties of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA    adduct. Cancer Res. 452440-4.-   13. Bello, M. S., de Besi, R., Rezzonico, R., Righetti, P. G., and    Casiraghi, E. 1994. Electroosmosis of polymer solutions in fused    silica capillaries. Electrophoresis 15:623-6.-   14. Bezrukov, S. M. Vodyanoy, I., and Parsegian, V. A. 1994.    Counting polymers moving through a single ion channel. Nature.    370:279.-   15. Bick, M. D. and Davisdon, R. L. 1974. Total substitution of    bromodeoxyuridine for thymidine in the DNA of a    bromodeoxyuridine-dependent cell line. Proc. Nat. Acad. Sci. USA    71:2082-2086.-   16. Bignold, L. P. 1987. A novel polycarbonate (Nuclepore) rembrane    demonstrates chemotaxis, unaffected by cherokinesis, of    polymorphonuclear leukocytes in the Boyden chamber. J. of    Immunological Methods. 105:275-280.-   17. Bloom, L. B., Turner, J., Kelman, Z, Beechem, J. M., O'Donnell,    M., and Goodman, M. F. 1996. Dynamics of loading the beta sliding    clamp of DNA polymerase III onto DNA. J. Biol. Chem. 271:30699-708.-   18. Bock, G., Hilchenbach, M., Schauenstein, K. and Wick, G. 1985.    Photometric analysis of antifading reagents for immunofluorescence    with laser and conventional illumination sources. J. of    Histochemistry and Cytochemistry 33:699-705.-   19. Bohmer, R. 1990. Cell division analysis using    bromodeoxyuridine-induced suppression of Hoechst 33258 fluorescence.    Methods in Cell Bology 18:173-84.-   20. Bruno, G., Capezzuto, P., and Madan, A. 1995. Plasma deposition    of amorphous silicon-based materials. Academic Press, Boston.-   21. Bustamante, C. 1991. Direct observation and manipulation of    single DNA molecules using fluorescence microscopy. Annu. Rev.    Biophys. Biophys. Chem. 20:415-46.-   22. Buurman, E. P., Sanders, R., Draaijer, A. Van Veen; J. J. F.,    Houpt, P. M., and Levine, Y. K. 1992. Fluorescence lifetime imaging    using a confocal laser scanning microscope. Scanning 14:155-59.-   23. Cantor, C. R., Mirzabekov. A, and Southern, E. 1992. Report on    the sequencing by hybridization workshop. Genomics. 13:1378-1383.-   24. Castro, A. and Shera, E. B. 1995. Single-molecule    electrophoresis. Anal. Chem. 67:3181-3186.-   25. Chang, H. T. and Yeung, E. S. 1995. Dynamic control to improve    the separation performance in capillary electrophoresis.    Electrophoresis. 16:2069-73.-   26. Chen, D. and Dovichi, N.J. 1996. Single-molecule detection in    capillary electrophoresis: molecular shot noise as a fundamental    limit to chemical analysis. Anal Chem. 68:690-696.-   27. Chu, G. Vollrath, D., and Davis, R. W. 1986. Separation of large    DNA molecules by contour-clamped homogeneous electric fields.    Science 234:1582.-   28. Church, G. M. and Kieffer-Higgins, S. 1988. Multiplex DNA    sequencing. Science 240:185-88.-   29. Clark, I., MacManus, J. P., Banville, D.; and Szabo, A. G. 1993.    A study of sensitized lanthanide luminescence in an engineered    calcium-binding protein. Anal. Biochem. 210:1-6.-   30. Clegg, R. M., Feddersen, B., Gratton, E., and Jovin, T. M. 1991.    Time-resolved imaging microscopy. Proc. SPIE—Int. Soc. Opt Eng.    1640:448-460.-   31. Clegg, R. M. 1992. Fluorescence resonance energy transfer and    nucleic acids. Methods in Enzymology. 211:353-379.-   32. Clegg, R. M., Murchie, A. I. H., Zechel, A., and    Lilley, D. M. J. 1993. Observing the helical geometry of    double-stranded DNA In solution by fluorescence resonance energy    transfer. Proc. Natl. Acad. Sci. USA. 90:2994-98.-   33. Clegg, R. M. 1995. Fluorescence resonance energy transfer. Curr.    Opin. Biotech. 6:103-110.-   34. Colson, P., Houssler, C. and Fredericq, E. 1974. Biochim.    Biophys. Acta 340:244-61.-   35. Crain, P. F. 1990. Mass Spectrom. Rev. 9:505-54.-   36. Crissnan, H. A. and Steinkamp, J. A. 1990. Detection of    bromodeoxyuridine-labeled cells by differential fluorescence    analysis of DNA fluorochromes. Methods in Cell Biology 33:199-206.-   37. Cundall, R. B. and Dale, R. E. (1983). Time-Resolved    Fluorescence Spectroscopy in Biochemistry and Biology. Plenum, New    York.-   38. Dash, J. G. 1975. Films on solid surfaces: the physics and    chemistry of physical adsorption. Academic Press, New York.-   39. Davis, L, Fairfield, F., Hammond, M., Harger, C., Jett, J., and    Keller, R. 1992. Rapid DNA sequencing based on single-molecule    detection. Los Alamos Science. 20:280-6.-   40. Davis, L. M., Fairfield, F. R., Harger, C. A., Jett, J. H.,    Keller, R. A., Hahn, J. H. Krakowski, L. A., Marrone, B. L,    Martin, J. C., Nutter, H. L., Ratliff, R. L., Shera, E. B.,    Simpson, D. J. and Soper, S. A. 1991. Genet. Anal. Tech. Appl.    8:1-7.-   41. de Jong, P. J., Yokobata, K., Chen, C., Lohman, F., Pederson,    L., McNinch, J. et al. 1989. Cytogenet. Cell Genet. 51:985.-   42. Ding, D. W., Rill, R. and Van Holde, K. E. 1972. Biopolymers    11:2109-2124.-   43. Dozier, J. 1988 HIRIS—The high resolution imaging spectrometer.:    Proc. SPIE—Recent Adv. Sensors, Radiometry Data Process. Remote    Sens. 924:10-22.-   44. Drmanac, R., Labat, I., Brukner, I., and Crkvenjakov, R. 1989.    Sequencing of megabase plus DNA by hybridization: theory of the    method. Genomics 4:114-128.-   45. Drmanac, R., Drmanac, S., Jarvis, J., and Labat, I. 1994.    Sequencing by hybridization. In Automated DNA Sequencing and    Analysis Techniques, J. Craig Ventor, Ed. Academic Press, London.-   46. Eigen, M. and Rigler, R. 1994. Sorting single molecules:    applications to diagnostics and evolutionary biotechnology. Proc.    Natl. Acad. Sci. USA. 91:5740-7.-   47. el-Deiry, W. S., et al., 1993. WAFI, a potential mediator of p53    tumor suppression. Cell 75:817-825.-   48. Faerber, P. and Scheit, K. H.1971. Chem. Ber. 104:456-460.-   49. Fairclough, R. H., and Cantor, C. H.1978. The use of    singlet-singlet energy transfer to study macromolecular assemblies.    Methods in Enzym. 347-79.-   50. Fan, F. F. and Bard, A. J. 1995. Electrochemical detection of    single molecules. Science 267:871-4.-   51. Fawcett, J. J., Longmire, J. L, Martin, J. C., Deaven, L. L.,    and Cram, L. S. 1994. Large-scale chromosome sorting. Methods in    Cell Biology 42:319-331.-   52. Feinberg, A. P., and Vogelsteln, B. 1983. A technique for    radiolabeling DNA restriction endonuclease fragments to high    specific activity. Anal. Biochem. 132:6-13.-   53. Feinberg, A. P. and Vogelsteln, B. 1984. A technique for    radiolabeling DNA restriction endonuclease fragments to high    specific activity. (Addendum). Anal. Biochem. 137:266-7.-   54. Ferreira, M. and Rubner, M. F. 1995. Molecular-level processing    of conjugated polymers. 1. layer-by-layer manipulation of conjugated    polymers. ACS 28:7107-7114.-   55. Ferrell, T., Allison, D., Thundat, T., and Warmack, R. 1997.    Scanning tunneling microscopy in sequencing of DNA. In Molecular    Biology and Biotechnology, R. A. Meyers, Ed. VCH Publishers, New    York.-   56. Förster, T. 1965. In Modem Quantum Chemistry, Vol. III    (Sinanoglu, O., Ed.), pp. 93-137, Academic Press, New York.-   57. Fou, A. C. and Rubner, M. F. 1995. Molecular-level processing of    conjugated polymers. 2. layer-by-layer manipulation of in-situ    polymerized p-type doped conducting polymers. ACS 28:7115-7120.-   58. Franklin, A. L. and Filion, W. G. 1985. A new technique for    retarding fading of fluorescence: DPX-BME. Stain Technology    60:125-35.-   59. Frey, M. W., Sowers, L. C., Millar, D. P., and    Benkovic, S. J. 1995. The nucleotide analog 2-aminopurine as a    spectroscopic probe of nucleotide incorporation by the Klenow    fragment of Escherichla coli polymerase I and bacteriophage T4 DNA    polymerase.-   60. Fu, T. J., Sanders, G. M., O'Donnell, M., and    Geiduschek, E. P. 1996. Dynamics of DNA-tracking by two    sliding-clamp proteins. EMBO J. 15:4414-22.-   61. Gadella, T. W. J., Jovin, T. M., and Clegg, R. M. 1993.    Fluorescence lifetime imaging microscopy (FLIM): spatial resolution    of microstructures on the nanosecond time scale. Biophysical    Chemistry. 48:221-239.-   62. Gains, H., Andersson, L, and Biberfeld, G. 1996. A new method    for measuring lymphocyte proliferation at the singlecell level in    whole blood cultures by flow cytometry. J. of Immunological Meth.    195:63-72.-   63. Garini, Y., Katzir, N., Cabib, D., and Buckwald, R. A. 1996.    Spectral bio-imaging. In Fluorescence Imaging Spectroscopy and    Microscopy (Wang, X.-F. and Herman, B., Eds.), John Wiley & Sons,    New York.-   64. Garland, P. B. and Moore, C. H.1979. Phosphorescence of    protein-bound eosin and erythrosin: a possible probe for    measurements of slow rotational motion. Biochem. J. 183, 561-572.-   65. Gawrisch, K, Han, K. H., Yang, J. S., Bergelson, L. D., and    Ferretti, J. A. 1993. Interaction of peptide fragment 828-848 of the    envelope glycoprotein of human immunodeficiency virus type I with    lipid bilayers. Biochemistry 32:31 12-18.-   66. Gebeychu, G. Rao, P. Y., SooChan, P., Simrs, D. A., and    Klevan, L. 1987. Novel biotinylated nucleotide-analogs for labeling    and colorimetric detection of DNA. Nucleic Acids Res. 15:4513-34.-   67. Gibson, T. J., Coulson, A. R., Sulston, J. E., and    Little. R. F. R. 1987a. Gene. 53:275.-   68. Gibson, T. J., Rosenthal, A., and Waterson, R. H.1987b. Gene    53:283.-   69. Gill, D. 1979. Inhibition of fading in fluorescence in    microscopy of fixed cells. Experientia 35:400-1.-   70. Gillam, I. C. and Tener, G. M. 1986. N⁴-(6-aminohexyl) cytidine    and -deoxycytidine nucleotides can be used to label DNA. Anal    Biochem. 157:199-207.-   71. Giloh, H. and Sedat, J. W. 1982. Fluorescence microscopy;    reduced photobleaching of rhodamine and fluorescein protein    conjugates by n-propyl gallate. Science 217:1252-1255.-   72. Glazer, A. N. and Mathies, R. A. 1997. Energy-transfer    fluorescent reagents for DNA analyses. Current Opinion in    Biotechnology 8:94-102.-   73. Glickman, B. W. 1985. Basic Life Sciences. 31:353-79.-   74. Glover, D. M. and Hames, B. D. 1995. DNA Cloning 3. IRL Press,    New York.-   75. Goodwin, P. M., Ambrose, W. P., Martin, J. C., and    Keller, R. A. 1995. Spatial dependence of the optical collection    efficiency in flow cytometry. Cytometry. 21:133-144.-   76. Gratton, E. and Llmkema, M. 1983. A continuously variable    frequency cross-correlation phase fluorometer with picosecond    resolution. Biophys. J. 44:315-325.-   77. Griep, M. A. 1995. Fluorescence recovery assay: a continuous    assay for processive DNA polymerases applied specifically to DNA    polymerase III holoenzyme. Anal Biochem. 232:180-9.-   78. Gudron, M., Eisinger, J., and Shulman, R. G. 1967. J. Chem.    Phys. 47:4077.-   79. Guilbault, G. G. 1973. (ed.) Practical fluorescence: theory,    methods and techniques. Marcel Dekker, New York.-   80. Gurrieri, S. Rizzarelli, E. Beach, D. and Bustamante, C. 1990.    Imaging of kinked configurations of DNA molecules undergoing    orthogonal field alternating gel electrophoresis by fluorescence    microscopy. Biochemistry 29:3396-3401.-   81. Haab, B. and Mathies, R. 1995. Single molecule fluorescence    burst detection of DNA fragments separated by capillary    electrophoresis. Anal. Chem. 67:3253-60.-   82. Hamada, S, and Fujilta, S. 1983. DAPI staining improved for    quantitative cytofluorometry. Histochemistry 79:219-26.-   83. Harding, J. D. and Keller, R. A. 1992. Single molecule detection    as an approach to rapid DNA sequencing. Trends Biotechnol: 10:55-57.-   84. Harris, C. M. and Kell, D. B. 1985. On the dielectrically    observable consequences of the diffusional motions of lipids and    proteins in membranes. 2. Experiments with microbial cells,    protoplasts and membrane vesicles. Eur. Biphysics. J. 13:11-24.-   85. Haughland. R. P. 1996. Handbook of Fluorescent Probes and    Research Chemicals. Molecular Probes, Eugene.-   86. Herendeen, D. R. and Kelly, T. J. 1996. DNA polymerase III:    running rings around the fork. Cell. 84:5-8.-   87. Herman, B., Wang, X. F., Periasamy, A., Kwon, S., Gordon, G.,    and Wodnicki, P. Fluorescence lifetime imaging in cell biology.    Proceedings of Optical Diagnostics of Living Cells and Biofluids    2678:88-97.-   88. Hogan, M., Dattagupta, N. and Crothers, D. M. 1978. Proc. Natl.    Acad. Sci. USA 75:195-199.-   89. Holtke, H.-J., Seibl, R., Burg, J., Mohlegger, K., and    Kessler, C. 1990. Non-radioactive labeling and detection of nucleic    acids: II. Optimization of the digoxigenin system. Mol. Gen.    Hoppe-Seyler 371:929-938.-   90. Holzwarth, A. R. 1995. Time-resolved fluorescence spectroscopy.    Methods in Enzymology. 246:335-361.-   91. Holzwarth, G., Whitconb, R. W., Platt, K. J., Crater, G. D., and    McKee, C. B. 1990. Velocity of linear DNA during pulsed-field gel    electrophoresis. In Electrophoresis of Large DNA Molecules (Lai, E.    and Birren, B. W., Eds). Cold Spring Harbor Laboratory Press, New    York.-   92. Holzwarth, G., McKee, C. B., Steiger, S., and Crater, G. 1987.    Transient orientation of linear DNA molecules during pulsed-field    gel electrophoresis. Biopolymes 28:1043.-   93. Houseal, T. W., Bustamante, C., Stump, R. F., and Maestre, M. F.    1989. Biophys. J. 56:507.-   94. Huff, J. C., Weston, W. L. and Wanda, K. D. 1982. Enhancement of    specific immunofluorescent findings with use of    para-phenylenediamine mounting buffer. J. of Investigative    Dermatology 78:449-50.-   95. Hung, S.C., Ju, J., Mathies, R. A., and Glazer, A. N. 1996.    Cyanine dyes with high absorption cross section as donor    chromophores in energy transfer primers. Anal. Biochem. 243:15-27.-   96. Ickhikawa, M., Iijima, T., and Matsumoto, G. 1993. Simultaneous    16, 384-site optical recording of neural activities in the brain. In    Brain Mechanisms of Perception and Memory: From Neuron to    Behanvior. T. Ono, L. R. Squire, M. E. Raichle, D. I. Perrett & M.    Fukuda (eds). Oxford University Press, NY.-   97. Ikehara, M., Tazawa, I., and Fukui, T. 1969a. Chem. Pharm. Bull.    (Tokyo). 17:1019-1024.-   98. Ikehara, M., Tazawa, I., and Fukui, T. 1969b. Biochemistry    8:736-43.-   99. Iijima, T., Ichikawa, M., & Matsumoto, G. 1989 Abstr. Soc.    Neurosci. 15:398.-   100. Janesick, J. (1980-91) Informal Notes, Jet Propulsion    Laboratory, California Institute of Technology, Pasedena, Calif.-   101. Jett, J., Keller, R., Martin, J., Marrone, B., Moyzis, R.,    Ratliff, R., Seitzinger, N., Shera, E., and Stewart, C. 1989.    High-speed DNA sequencing: an approach based upon fluorescence    detection of single molecules. J. of Bio. Structure & Dynamics    7:301-9.-   102. Jett et al. 1995. U.S. Pat. No. 5,405,747.-   103. Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G.,    Goodwin, D. & Holborow, E. J. 1982. Fading of immunofluorescence    during microscopy: a study of the phenomenon and its remedy. J. of    Immunological Methods 55:231-242.-   104. Johnson, G. D. and Nogueira Araujo, G. M. de C. 1981. A simple    method of reducing the fading of Immunofluorescence during    microscopy. J. of Immunological Methods 43:349-50.-   105. Ju, J., Glazer, A. N., and Mathies, R. A. 1996a. Cassette    labeling for facile construction of energy transfer fluorescent    primers. Nucleic Acids Res. 24:1144-8.-   106. Ju, J., Glazer, A. N., and Mathies, R. A. 1996b. Energy    transfer primers: a new fluorescence labeling paradigm for DNA    sequencing and analysis. Nature Medicine. 2:246-9.-   107. Jumppanen. J. H. and Riekkola, M. L. 1995. Influence of    electrolyte composition on the effective electric field strength in    capillary zone electrophores. Electrophoresis 16:1441-4.-   108. Kasianowicz, J. J., Brandin, E., Branton, D.; and    Deamer, D. W. 1996. Characterization of individual polynucleotide    molecules using a membrane channel. Proc. Natl. Acad. Sci. USA.    93:13770-3.-   109. Kato, H. and Yoshida, T. H.1970. Nondisjunction of chromosomes    in a synchronized cell population initiated by reversal of colcemid    inhibition. Expl. cell. Res. 60:459-64.-   110. Keough, T., Baker, T. R., Dobson, R. L. M., Lacey, M. P. Riley,    T., Hasseltield, J., and Hesselberth, P. E. 1993. Rapid Commun. Mass    Spectrom. 7:195-200.-   111. Klnjo, M. and Rigler, R. Ultrasensitive hybridization analysis    using fluorescence correlation spectroscopy. Nucleic Acids Research.    23:1795-1799.-   112. Kirk, W. R., Wessels, W. S., and Prendergast, F. G. 1993.    Lanthanide-dependent perturbations of luminescence in    indolylethylenediaminetetraacetic acid-lanthanide chelate. J. Phys.    Chem. 97:10326-10340.-   113. Klenchin, V. A., Sukharev, S. I., Serov, S. M.,    Chernomordik, L. V., and Chizmadzhev, YuA. 1991. Electrically    induced DNA uptake by cells is a fast process involving DNA    electrophoresis. Biophysical J. 60:804-11.-   114. Kochetkov, N. K, Budowsky, and Shibaev, V. N. Proc. Nucl. Acid    Chem., 1:500-2.-   115. Konuma, M. 1992. Film Deposition by plasma techniques.    Springer-Verlag, New York.-   116. Korchemnaya, E. K., Ermakov, A. N., Bochkova, L. P. 1978. J.    Anal Chem USSR (Eng. Transl.) 33:625.-   117. Kornberg, A., and Baker, T. A. 1991. DNA Replication W. H.    Freeman, New York.-   118. Lakowicz, J. R. and Szmacinski, H.1996. Imaging applications of    time-resolved fluorescence spectroscopy. In Fluorescence Imaging    Spectroscopy and Microscopy (Wang, X. F. and Herman, B. Eds). John    Wiley & Sons, New York.-   119. Lakowicz, J. R. and Berndt, K. W. 1991. Lifetime-selective    fluorescence imaging using an RF phase-sensitive camera. Rev. Sci.    Instru. 62:3653.-   120. Lang, R. A., Robins, R. K., and Townsend, L. B. 1968. Synthetic    Procedures in Nucleic Acid Chemistry. 1:228. Ed, Zorbach, W. W. and    Tipson, R. S. John Wiley & Sons, New York.-   121. Langer, P. R., Waldrop, A. A., and Ward, D.C. 1981. Enzymatic    synthesis of biotin-labeled polynucleotides: novel nucleic acid    affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78-6633-6637.-   122. Lee, K. B., Matsuoka, K, Nishimura; S, and Lee, Y. C. 1995. A    new approach to assay endo-type carbohydrases: bifluorescent-labeled    substrates for glycoamidases and ceramide glycanases. Anal. Biochem.    230:31-6.-   123. Lee, L. G., Connell, C. R., Woo, S. L., Cheng, R. D.,    McArdle, B. F., Fuller, C. W., Halloran. N. D., and Wilson, R. K.    Nucleic Acids Res. 20:2471-2483.-   124. Lee, S. P., Porter, D., Chirikjian, J. G., Knutson, J. R., and    Han, M. K. 1994. A fluorometric assay for DNA cleavage reactions    characterized with BamHI restriction endonuclease. Anal. Biochem.    220:377-383.-   125. Lee, S. P., Censullo, M. L, Kim, H. G., Knutson, J. R., and    Han, M. K. 1995. Characterization of endonucleolytic activity of    HIV-1 integrase using a fluorogenic substrate. Anal. Biochem.    227:295-301.-   126. Lee, Y—H, Maus, R. G., Smith, B. W. and    Winefordner, J. D. 1994. Laser-induced fluorescence detection of a    single molecule in a capillary. Anal. Chem. 66:4142-9.-   127. Lewotsky, K. 1994. Hyperspectral imaging: evolution of imaging    spectroretry. SPIE OE/Rep., November:1-3.-   128. Little, D. P., Chorush, R. A., Speir, J. P., Senko, M. W.,    Kelleher, N. L., and McLalferty, F. W. 1994. J. Am. Chem. Soc.    116:4893-897.-   129. Livak, K. J. 1997. Quantitation of DNA/RNA using real-time PCR    detection. PE Applied Biosystems, Fostcr City, Calif.-   130. Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T. et    al., 1996. Expression monitoring by hybridization to high-density    oligonucleotide arrays. Nature Biotechn. 14:1675-1680.-   131. Loros, J. J., Denome, S. A., and Dunlap, J. C. 1989. Molecular    cloning of genes under control of the circadian clock in Neurospora.    Science 243:385-388.-   132. Marcus, P. I. and Robbins, E. 1963. Viral inhibition in the    metaphase-arrest cell. Proc. Natl. Acad. Sci. USA 50:1156-64.-   133. Marra, M., Weinstock, L. A., and Mardis, E. R. 1996. End    sequence determination from large insert clones using energy    transfer fluorescent primers. Genome Research 6:1118-22.-   134. Martin, I, Dubols, M-C., Defrise-Quertain, F., Saermark, T.,    Bumy, A., Brasseur, R., and Ruysschaert, J-M. 1994. Correlation    between fusogenicity of synthetic modified peptides corresponding to    the NH₂-terminal extremity of simian immunodeficiency virus gp32 and    their mode of insertion into the lipid bilayer: an infrared    spectroscopy study. J. Virol. 68:1139-48.-   135. Mason, W. T. 1992. Fluorescent and Luminescent Probes for    Biological Activity. Harcourt Brace & Company, Cambridge, UK.-   136. Matayoshi, E. D., Wang, G. T., Kraft, G. A., and    Erickson, J. 1990. Novel fluorogenic substrates for assaying    retroviral proteases by resonance energy transfer. Science. 247:954.-   137. Matsumoto, B. 1993. Cell biological applications of confocal    microscopy. Methods in Cell Biology. 38:86.-   138. Matsumoto, S., Morikawa, K., and Yangida, M. 1981. Light    microscopic structure of DNA in solution studied by the    4′,6-diamidino-2-phenylindole staining method. J. Mol. Biol.    152:501-516.-   139. Maurizi, M. R., Kasprzyk, P. G., and Ginsberg, A. 1986.    Distances between active site probes in glutamine synthetase from    Escherichia coli fluorescence energy transfer in free and in stacked    dodecamers. Biochem 25:141-151.-   140. Mautner, H. G. 1956. J. Am. Chem. Soc. 78:5293.-   141. Maxam, A. M. and Gilbert, W. 1977. A new method for sequencing    DNA. Proc. Natl. Acad. Sci. USA. 74:5604.-   142. Maymon, W., and Neeck, S. P. 1988. Optical system design    alternatives for the Moderate-Resolution Imaging Spectrometer Tilt    (MODIS-T) for the Earth Observing System (EoS). Proc. SPIE-Recent    Adv. Sensors, Radiometry Data Process Remote Sens. 924:10-22.-   143. McGown, L. B. 1989. Fluorescence lifetime filtering. Anal.    Chem. 61:839 A-847A.-   144. Meffert, R. and uose, K. 1988. Uv-induced cross-linking of    proteins to plasmid pER322 containing 8-azidoadenine    2′-deoxyribonucleotides. FEBS Lets. 239:190-4.-   145. Meertz, J., Xu, C. and Webb, W. W. 1995. Single-molecule    detection by two-photon-excited fluorescence. Optics Letters    20:253234.-   146. Meinkoth, J. and Wahl, G. M. 1987. Nick translation. Methods in    Enzymology 152:91-94.-   147. Menter, J. M., Hurst, R. E. and West, S. S. 1979.    Photochemistry of heparin-acridine orange complexes in solution.    Photochemistry and Photobiology. 27:629-33.-   148. Menter, J. M., Golden, J. F. & West, S. S. 1978. Kinetics of    fluorescence fading of acridine orange-heparin complexes in    solution. Photochemistry and Photobiology 27:629-633.-   149. Miki, M., O'Donoghue, S. I., and Dos Remedios, C. G. 1992.    Structure of actin observed by fluorescence resonance energy    transfer spectroscopy. J. Muscle Res. Cell Motil. 13:132.-   150. Miki, M. and Iio, T. 1993. Kinetics of structural changes of    reconstituted skeletal muscle thin filaments observed by    fluorescence resonance energy transfer. J. Biol. Chem.    268:7101-7106.-   151. Mitchinson, J. M. 1971. The Biology of the Cell Cycle.    Cambridge University Press, London.-   152. Moore, D. P., Schellman, J. A., and Baase, W. A. 1986. The    orientation, relaxation and reptation of DNA in orthogonal field,    alternately-pulsed field gel electrophoresis: a linear dichroism    study.” Biopphys. J. 49:130a.-   153. Morikawa, K., and Yangida, M. 1981. J. Biochem. 89:693.-   154. Morosanu, C. E. 1990. Thin films by chemical vapour deposition.    Elsevier, New York.-   155. Morgan, C. G., Mitchell, A. C., and Murray, J. G. 1990.    Nanosecond time-resolved fluorescence microscopy: principle and    practice. Trans. R. Microsc. Soc. (Micro '90) 463-466.-   156. Nakashima, M., Yamada, S., Shiono, S., and Maeda, M. 1992. IEEE    Trans. Biomed. Engng. 39:26-36.-   157. Naktinis, V., Turner, J., O'Donnell, M. 1996. A molecular    switch in a replication machine defined by an internal competition    for protein rings. Cell. 84:127-45.-   158. Negishl, K., Matsumoto, K., Bessho, T., Tada, F., and Hayatsu,    H.1988. In vitro mutagenesis by incorporation of    N4-aminodcoxycytidine 5′-triphosphatc. Nucleic Acids Symposium    Series. 19:33-6.-   159. Newman, J., Swiney, J. L, Day, L. A. 1977. J. Mol. Biol.    110:119-46.-   160. Nicolas, V., Nefussi, J. R., Collin, P., and Forest, N. 1990.    Effects of acidic fibroblast growth factor and epidermal growth    factor on subconfluent fetal rat calvaria cell cultures: DNA    synthesis and alkaline phosphatase activitiy. Bone and Mineral.    8:145-56.-   161. Nguyen, D.C. and Keller, R. A. Detection of single molecules of    phycoerythrin in hydrodynamically focused flows by laser-induced    fluorescence. Anal. Chem. 59:2158-2161.-   162. Nie, S., Chiu, D. T., and Zare, R. N. 1994. Probing individual    molecules with confocal fluorescence microscopy. Science.    266:1018-21.-   163. Oida, T., Sato, Y., and Kusumi, A. 1993. Fluorescence lifetime    imaging microscopy (filmscopy): methodology development and    application to studies of endosome fusion in single cells.    Biophys. J. 64:676-685.-   164. Onrust, R., and O'Donnell, M. 1993. J. Biol. Chem.    268:11766-72.-   165. Pap, E. H. W., Bastiaens, P., Borst, J. W., van den Berg, P.,    van Hoek, A., Snoek, G., Wirtz, K, and Visser, A. 1993. Quantitation    of the interaction of protein kinase C with diacylglycerol and    phosphoinostitides by time-resolved detection of resonance energy    transfer. Biochemistry 32:13310-17.-   166. Paul, W. Professor of Physics, Harvard University.-   167. Pauleau, Y. 1995. Materials and processes for surface and    interface engineering. Kluwer Academic Publishers, Boston.-   168. Paz-Elizur, T., Skaliter, R., Blumenstein, S., and    Uvneh, Z. 1996. Beta*, a UV-Inducible smaller form of the beta    subunit sliding clamp of DNA polymerases III of Esherichia coli. I.    Gene expression and regulation. J. Biol. Chem. 271:2482-90.-   169. Peck, K., Stryer, L, Glazer, A. N. and Mathies, R. A. 1989.    Single-molecule fluorescence detection: autocorrelation criterion    and experimental realization with phycoerythrin. Proc. Natl. Acad.    Scl. USA 86:4087-4091.-   170. Periasamy, A., Siadat-Pajouh, M., Wodnick, P., Wang, X-F., and    Herman, B. Time-gated fluorescence microscopy for clinical imaging.    Microscopy and analysis. 3:334.-   171. Periasamy, A. & Herman, B. 1994. Computerized fluorescence    microscopic vision in the biomedical science, J. of    Computer-Assisted Microscopy 6:1-26.-   172. Picciolo, G. L. and Kaplan, D. S. 1984. Reduction of fading of    fluorescent reaction product for microphotometric quantitation.    Advances in Applied Microbiology 30:197-234.-   173. Pillai, V. N. 1980. Photoremovable protecting groups in organic    synthesis. Synthesis. 1980:1.-   174. Platt, J. L. and Michael, A. F. 1983. Retardation of fading and    enhancement of intensity of immunofluorescence by    p-phenylenediamine. J. of Histochemistry and Cytochemistry    31:840-42.-   175. Poot, M. and Hoehn, H.1990. Cell cycle analysis using    continuous bromodeoxyuridine labeling and Hoeschst 33258-ethidium    bromide bivariate flow cytometry. Methods in Cell Biology 33:185-98.-   176. Porter, G. (Ed.) 1967. Reactivity of the photoexcited organic    molecule. Interscience, New York.-   177. Pringsheim, P. 1963. Fluorescence and phosphorescence. John    Wiley, New York.-   178. Priore, D. R. C. and Allen. F. S. 1979. Biopolymers    18:1809-1820.-   179. Purmal, A. A., Kow, Y. W., and Wallace, S. S. 1994.    5-Hydroxyprimidine deoxynucleoside triphosphates are more    efficiently incorporated into DNA by exonuclease-free Klenow    fragment than 8-oxopurine deoxynucleoside triphosphates. Nucleic    Acids Res. 22:3930-5.-   180. Purcell, E. M. 1985. Electricity and Magnetism, Vol. 2    McGraw-Hill, New York.-   181. Qu, D., et al., 19996. A role for melanin-concentrating hormone    in the central regulation of feeding behaviour. Nature 380:243-247.-   182. Rahn, R. 0., Schulman, R. G., and Longworth, J. W. 1965. Proc.    Natl. Acad. Sci. USA 53:893.-   183. Rampino, N.J. and Chrambach, A. 1990. Apparatus for gel    electrophoresis with continuous monitoring of individual DNA    molecules by video epifluorescence microscopy. Anal. Biochem.    194:278-283.-   184. Rao, P. N. 1968. Mitotic synchrony in mammalian cells treated    with nitrous oxide at high pressures. Science 160:774-6.-   185. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. 1977.    Labeling deoxyribonucleic acid to high specific activity in vitro by    nick translation with DNA polymerase I. J. Mol. Biol. 113:237-51.-   186. Reddick, R. C., Warmack, R. J., and Ferrell, T. L. 1989. Phys.    Rev. B 39:767-770.-   187. Rodgers, M. A. J. and Firey, P. A. 1985. Photochem. Photobiol.    42:613.-   188. Ronaghi, M., Karamohamed, S., Pettersson, B., Uhln, M., and    Nyrn, P. 1996. Real-time DNA sequencing using detection of    pyrophosphate release. Anal. Biochem. 242:84-89.-   189. Ross, P. D. and Scruggs, L. 1964. Biopolymers. 2:231-6.-   190. Rost, F. W. D. 1991. Quantitative Fluorescence Microscopy.    Cambridge University Press, Cambridge.-   191. Roychoudhury, R., Tu, C.—P-D., and Wu, R. 1979. Influence of    nucleotide sequence adjacent to duplex DNA termini on 3′-terminal    labeling by terminal transferase. Nucleic Acids Res. 6:1323-1333.-   192. Saha, A. K., Kross, K., Kloszewski, E. D., Upson, D. A.,    Toner, J. L, Snow, R. A., Black, C. D. V., and Desai, V. C. 1993.    Time-resolved fluorescence of a new europium chelate complex:    demonstration of highly sensitive detection of protein and DNA    samples. J. Am. Chem. Soc. 115:11032-33.-   193. Sahota, R. S, and Khaledi, M. G. 1994. Nonaqueous capillary    electrophoresis. Anal. Chem. 66:1141-6.-   194. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G.    T., Erlich, H. A., and Amheim, N. 1985. Enzymatic amplification of    beta-globin genomic sequences and restriction site analysis for    diagnosis of sickle cell anemia. Science 230:1350-1354.-   195. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J.,    Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. 1988.    Primer-directed enzymatic amplification of DNA with a thermostable    DNA polymerase. Science 239:487-91.-   196. Sanger, F., Nicklen, A., and Coulson, A. R. 1977. DNA    sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.    USA. 74:5463-7.-   197. Sase, I., Miyata, H., Corrie, J., Cralk, J, and Kinosita,    Jr., K. K. 1995. Real time imaging of single fluorophores on moving    actin with an epifluorescence microscope. Biophys. J. 69:323-8.-   198. Scheit, K. H. 1980. Nucleotide Analogs: Synthesis and    Biological Function. John Wiley & Sons, New York.-   199. Schellman, J. A., and Jensen, H. P. 1987. Optical spectroscopy    of oriented molecules. Chem. Rev. 87:1359.-   200. Schott, J. R. 1989. Remote sensing of the Earth: A synoptic    view. Phys Today September: 72-79.-   201. Schwartz, D.C. and Koval, M. 1989. Conformational dynamics of    individual DNA molecules during gel electrophoresis. Nature    338:520-522.-   202. Selvin, P. R., Rana, T. M., and Hearst, J. E. 1994.    Luminescence resonance energy transfer. J. Am. Chem. Soc.    116:6029-30.-   203. Selvin, P. R. 1995. Fluorescence resonance energy transfer.    Methods in Enzymology. 246:300-334.-   204. Shera, E. B., Seitzinger, N. K., Davis, L. M., Keller, R. A.,    and Soper, S. A. 1990. Detection of single fluorescent molecules.    Chem. Phys. Letts. 174:553-57.-   205. Shlkmus, M. L., Guaglianone, P., and Herman, T. M. 1986.    Synthesis and characterization of blotin-labeled nucleotide analogs.    DNA. 5:247-55.-   206. Skaliter, R., Bergstein, M., and Livneh, Z 1996. Beta*, a    UV-inducible shorter form of the beta subunit of DNA polymerase III    of Escherichia coli. II. Overproduction, purification, and activity    as a polymerase processivity clamp. J. Biol. Chem. 271:2491-6.-   207. Skoog, D. A., West, D. M., and Holler, F. J. 1992. Analytical    Chemistry. Saunders College Publishing, New York.-   208. Smirnov, I. P., Roskey, M. T., Juhasz, P., Takach, E. J.,    Martin, S. A., and Half, L. A. 1996. Sequencing oligonucleotides by    exonuclease digestion and delayed extraction matrix-assisted laser    desorption ionization time-of-flight mass spectrometry. Anal    Biochem. 238:19-25.-   209. Smith, D. P., Shleh, B. H., and Zuker, C. S. 1990. Isolation    and structure of an arrestin gene from Drosophila. Proc. Natl. Acad.    Sci. USA 87:1003-1007.-   210. Smith, S. B., Burrieri, S., and Bustamante, C. 1990.    Fluorescence microsocpyand computer simulations of DNA molecules in    conventional and pulsed-field gel. electrophoresis. In    Electrophoresis of Large DNA Molecules (Lai, E. and Birren, B. W.,    Eds). Cold Spring Harbor Laboratory Press, New York.-   211. Smith, S. B., Aldridge, P. K., and Callis, J. B. 1989.    Observation of individual DNA molecules undergoing gel    electrophoresis. Science 243:203-206.-   212. Smith, L. M., Sanders, J. Z. Kaiser, R. J., Hughes, P., Dodd,    C., Connell, C. R., Heiner, C., Kent, S. B., and Hood, L. E. 1986.    Fluorescence detection in automated DNA sequence analysis. Nature.    321:674-9.-   213. Sober, H. A. Ed. 1970. Handbook of Biochomistry, 2nd ed. The    Chemical Rubber Co., Cleveland.-   214. Soper, S. A., Davis, L. M. and Shera. E. B. 1992. Single    molecule spectroscopy in solution. Los Alamos Science 20:286-96.-   215. Spatz, W. B. and Grabig, S. 1983. Reduced fading of fast blue    fluorescence in the brain of the guinea-pig by treatment with    sodium-nitroprusside. Neuroscience Letters 38:1-4.-   216. Spohr, R. 1990. Ion tracks and microtechnology: principles and    applications Vieweg, Braunschweig.-   217. Steinberg, I. Z. 1971. Annu. Rev. Biochem. 40:83.-   218. Steiner, R. F. and Weinryb, I. 1971. Excited States of Proteins    and Nucleic Acids. Plenum Press, New York.-   219. Stewart, J. E., Hahn, G. M., Parker, V., and    Bagshaw, M. A. 1968. Chinese hamster cell monolayer cell cultures.    Exp. Cell Res. 49:293-299.-   220. Stuart, R: V. 1983. Vacuum technology, thin films, and    sputtering: an introduction. Academic Press. New York.-   221. Stryer, L. 1978. Annual Review of Biochem. 47:819.-   222. Stubblefield, E. 1968. Synchronization methods for mammalian    cell cultures. In Methods in Cell Physiology. Ed. by D. M. Prescott.    3:25-43. Academic Press, New York.-   223. Sturm, J. and Weill, G. 1989. Direct observation of DNA chain    orientation and relaxation by electric birefringence: implications    for the mechanism of separation during pulsed-field gel    electrophoresis. Phys. Rev. Lett. 62:1484.-   224. Taliani, M., Bianchi, E., Narjes, F., Fossatelli, M., Urbani,    A., Steinkuler, C., De Francesco, R., and Pessi, A. 1996. A    continuous assay of hepatitis C virus protease based on resonance    energy transfer dipeptide substrates. Anan. Biochem. 240:60-7.-   225. Taylor, D. L. and Salmon, E. D. 1989. Methods in Cell Biol.    29:207-37.-   226. Taylor, D. L, Reidler, J., Spudich, J. A., and Stryer, L 1981.    Detection of actin assembly by fluorescence energy transfer. J. Cell    Biol. 89:363.-   227. ter Beest, M. and Hoekstra, D. 1993. Interaction of myelin    basic protein with artificial membranes. Parameters governing    binding, aggregation and dissociation. Eur. J. Biochem. 211:689-96.-   228. Theil, E. 1997. Automation in genome research. In Molecular    Biology and Biotechnology, R. A. Meyers, Ed. VCH Publishers, New    York.-   229. Thomas, R. S., Shimkunas, A. R., and Manger, P. E. 1992.    Sub-nanosecond intensifier gating using heavy and mesh cathode    underlays. Proc. Int. Congr. High Speed Photo. Photon 19th. 1984.-   230. Tian, R. and Rodgers, M. A. J. (1991). Time-resolved    fluorescence microscopy. In New Techniques in Optical Microscopy and    Spectrophotometry (R. J. Chemy, Ed.), pp. 312-351. CRC Press, Boca    Raton, Fla.-   231. Tinoco, I., Sauer, K., and Wang, J. C. 1995. Physical Chemistry    Prentice Hall, Englewood Cliffs.-   232. Tu, C.—P-D., and Cohen, S. 1980 3′-End labeling of DNA with    α-³²P cordycepin-5′-triphosphate. Gene 10:177-83.-   233. Uchiyama, H. Hirano, K., Kashiwasake-Jibu, M., and    Taira, K. 1996. Detection of undegraded oligonucleotides in vivo by    fluorescence resonance energy transfer. J. Biol. Chem. 271:380-84.-   234. Udenfriend, S. 1962. Fluorescence assay in biology and    medicine. Academic Press, New York and London.-   235. Ueda, T., Miura, K., Imazawa, K., and Odajima, K. 1974. Chem.    Pharm. Bull. (Tokyo). 22:2377-2382.-   236. Valiev, K. A. 1992. The physics of submicron lithography.    Plenum Press, New York.-   237. Valnes, K. and Brandtzaeg, P. 1985. Retardation of    immunofluorescence fading during microscopy. J. of Histochemistry    and Cytochemistry 33:755-761.-   238. van Huist, N. F., Moers, M. H. P., Noordman, 0. F. J., Tack, R.    G., Segerink, F. B., and Boger, B. 1993. Appl. Phys. Lett. 62:461.-   239. van de Ven, M., and Gratton, E. 1992. Time-resolved    fluorescence lifetime imaging. In Optical Microscopy: Emerging    Methods and Applications (B. Herman, and J. J. Lemaster, Eds.),    373-402. Academic Press, San Diego.-   240. Vaughan, W. M. and Weber, G. 1970. Oxygen quenching of    pyrenebutyric acid fluorescence in water. A dynamic probe of the    microenvironment. Biochemistry 9:464-473.-   241. Waggoner, A. 1995. Covalent labeling of proteins and nucleic    acids with fluorophores. Methods in Enzymology 246:362-373.-   242. Wang et al., 1995. Discovery of adrenomedullin in rat ischemic    cortex and evidence for its role in exacerbating focal brain    ischemic damage. Proc. Natl. Acad. Sci. USA 87:1003-1007.-   243. Wang, S. Y. 1976. Photochemistry and Photobiology of Nucleic    Acids. Academic Press, New York.-   244. Wang, X. F., Periasamy, A., Wodnicki, P., Gordon, G. W., and    Herman, B. 1996. Time-resolved fluorescence lifetime imaging    microscopy: Instrumentation and biomedication applications. In    Fluorescence Imaging Spectroscopy and Microscopy (Wang, X. F. and    Herman, B. Ed). John Wiley & Sons, Inc., New York.-   245. Wang, X. F., Periasamy, A., Wodnicki, P., Gordon, G. W., and    Herman, B. 1996. Time-resolved fluorescence lifetime imaging    microscopy: instrumentation and biomedical applications. Chemical    Analysis 137:313-350.-   246. Wang, X. F., Kitajima, S., Uchida, T., Coleman, D. M., and    Minami, S. 1990. Time-resolved fluorescence microscopy using    multichannel photon counting. Appl. Spectrosc. 44:25.-   247. Wang, Y., Wallin, J. M., Ju, J., Sensabaugh, G. F., and    Mathies, R. A. 1996. High-resolution capillary array electrophoretic    sizing of multiplexed short tandem repeat loci using energy-transfer    fluorescent primers. Electrophoresis. 17:1485-90.-   248. Wang, Y., Ju, J., Carpenter, B. A., Atherton, J. M.,    Sensabaugh, G. F., and Mathies, R. A. 1995. Rapid sizing of short    tandem repeat alleles using capillary array electrophoresis and    energy-transfer fluorescent primers. Anal. Chem. 67:1197-203.-   249. Ward, D.C. and Reich, E. 1969. Fluorescence studies of    nucleotides and polynucleotides. 244:1228-1237.-   250. Weimann, S., Rupp, T., Zimmermann, J., Voss, H., Schwager, C.,    and Ansorge, W. 1995. Primer design for automated DNA sequencing    utilizing T7 DNA polymerase and internal labeling with    fluorescein-15-dATP. BioTechniques 18:688-97.-   251. Wilkinson, J. Q., Lanahan, M. B., Conner, T. W., and    Klee, H. J. 1995. Identification of mRNAs with enhanced expression    in ripening strawberry fruit using polymerase chain reaction    differential display. Plant Mol. Biol. 27:1097-1108.-   252. Wittwer, C. T. Herman, M. G., Moss, A. A., and    Rasmussen, R. P. 1997. Continuous fluorescence monitoring of rapid    cycle DNA amplification. Biotechniques. 22:130-1, 134-8.-   253. Wooley, A. T. and Mathies, R. A. 1995. Ultra-high-speed DNA    sequencing using capillary electrophoresis chips. Anal. Chem.    67:3676-3680.-   254. Woronicz, J. D., Calnan, B.; Ngo, V., and Winoto, A. 1994.    Requirement for the orphan steroid receptor Nur77 in apoptosis of    T-cell hybridomas. Nature 367:277-281.-   255. Wu, H. M., Dattagupta, N. and Crothers, D. M. 1981. Proc. Natl.    Acad. Sci. USA 78:6808-6811.-   256. Wu, P. G. and Brand, L. 1994. Resonance energy transfer:    methods and applications. Anal. Biochem. 218:1-13.-   257. Wunderlich, F. and Peyk, D. 1969. Antimritotic agents and    macronuclear division of ciliates. II. Endogeneous recovery from    colchicine and colcemid-a new method of synchronization in    Tetrahymena pyriformis GL Expl. Cell Res. 57:142-4.-   258. Yanagida, M., Hiraoka, Y., and Katsura, I. 1983. Cold Spring    Harbor Symp. Biol. 47:177-87.-   259. Yamaoka, K. and Matsuda, K 1981. Macromolecules 14:595-601.-   260. Yamaoka, K. and Chamey, E. 1973. Macromolecules 6:66-76.-   261. Vane, G., Chrien, T. G., Reimer, J. H., Green, R. O., and    Conel, J. E. 1988. Comparison of laboratory calibrations of the    Airborne Visible/infrared imaging Spectrometer (AVIRIS) at he    beginning and end of the first flight season. Proc. SPIE—Recent Adv.    Sensors, Radiometry Data Process. Remote Sens. 924:168-178.-   262. Zweig, A. 1973. Photochemical generation of stable fluorescent    compounds (photofluorescence). Pure and Applied Chemistry    33:389-410.

1.-160. (canceled)
 161. A system for analyzing biological molecules,comprising: a device having a plurality of passageways; a molecularmotor in communication with at least one of the passageways, themolecule motor configured to receive a biological molecule, and furtherconfigured to move the biological molecule at least partially throughthe at least one passageway.
 162. The system of claim 161, wherein theat least one passageway is a nanopore.
 163. The system of claim 161,wherein the at least one passageway is a nanochannel.
 164. The system ofclaim 161, wherein each of the plurality of passageways is incommunication with a molecular motor, and each molecular motor isconfigured to receive a distinct biological molecule, and each molecularmotor is configured to move the distinct biological molecules throughdistinct passageways.
 165. The system of claim 164, wherein thepassageways are nanopores.
 166. The system of claim 161, wherein themolecular motor is a polymerase.
 167. The system of claim 161, whereinthe molecular motor is a helicase.
 168. The system of claim 161, whereinthe biological molecule is DNA.
 169. The system of claim 168, whereinthe biological molecule is single-stranded DNA.
 170. The system of claim161, wherein the molecular motor is configured to linearly thread thebiological molecule through the at least one passageway.
 171. The systemof claim 161, further comprising a station, the station configured toproduce a detectable signal as the biological molecule moves relative tothe station.
 172. The system of claim 171, wherein the station undergoesa physical change as the biological molecule moves relative to thestation, the physical change producing the detectable signal.
 173. Thesystem of claim 171, wherein the detectable signal is a conductancemeasured across the passageway.
 174. The system of claim 171, whereinthe detectable signal is a resistance measured across the passageway.175. The system of claim 171, wherein the station is at least a portionof the at least one passageway.
 176. The system of claim 171, whereinthe biological molecule is single-stranded DNA, and the detectablesignals relate to a nucleotide sequence of the single-stranded DNA. 177.The system of claim 161, wherein the molecular motor ratchets thebiological molecule through the at least one passageway.
 178. A DNAsequencing system, comprising: a plurality of molecular motors, eachmolecular motor corresponding to a distinct pore, each molecular motorconfigured to receive a distinct DNA molecule, and further configured tothread the distinct DNA molecule through the corresponding distinctpore; and at least one detector configured to detect at least one signalfrom at least one pore as the distinct DNA molecule moves relative tothe distinct pore, the at least one signal relating to a sequence ofnucleotides of the distinct DNA molecule.
 179. The system of claim 178,wherein the DNA molecule is a single-stranded DNA molecule, and themolecular motor is a polymerase.
 180. A biological analysis system,comprising: a plurality of molecular motors, each molecular motorcorresponding to a distinct pore of a device, each molecular motorconfigured to receive a distinct DNA molecule, and further configured tothread the distinct DNA molecule through the corresponding distinctpore.